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LIPID PRODUCTION

INCORPORATION BY CROSS-REFERENCE

This application claims priority from Australian provisional patent application number 2010905431 (AU 2010905431) filed on 9 December 2010 and Australian provisional patent application number 2011902937 (AU 2011902937) filed on 22 July 2011. The entire contents of AU 2010905431 and AU 2011902937 are incorporated herein by cross-reference.

TECHNICAL FIELD

The invention relates to methods for producing lipids and in particular methods for producing glycerolipids suitable for use in generating biofuels. The invention further relates to genetically modified plants and microorganisms in which the production of glycerolipids/oil is increased.

BACKGROUND

Biofuels (e.g. biodiesel) may be derived from lipid-containing vegetable oils such as soybean oil and palm oil. However, given the current food crisis alternative sources of vegetable oils for biofuel production need to be identified to reduce the reliance on edible plants. Photosynthetic microorganisms (e.g. microalgae) are a promising alternative for the production of the vegetable oils as they possess high lipid content and do not require arable land. The direct use of vegetable oils derived from plants and microorganisms such as algae in currently available diesel engines is impractical predominantly due to issues concerning viscosity, volatility and acid contamination. Therefore, lipids in vegetable oils derived from these sources must be processed to acquire properties similar to that of petrodiesel. A commonly used technique of producing biodiesel is the transesterification of triacylglycerols (TAGs), the primary lipid component of vegetable oils. Both the yield and quality of vegetable oil TAGs from the source organism utilised are critical factors in biodiesel production. For example, it is preferable to produce TAGs with shorter and saturated acyl chains because the resulting fatty acids possess more favourable properties as transportation fuels.

There is a need for improved methods of generating lipids suitable for biofuel production. A need also exists for plants and microorganisms with characteristics that improve the yield and/or quality of lipids suitable for biofuel production. ,

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SUMMARY OF THE INVENTION

In a first aspect, the invention provides a method for increasing triacylglycerol production in a cell, the method comprising modifying the cell to increase phosphatidic acid production.

In a second aspect, the invention provides a method for increasing the energry-storage potential of a cell, the method comprising modifying the cell to increase phosphatidic acid production.

In one embodiment of the second aspect, the method comprises fusing multiple lipid droplets within the cell.

In one embodiment of the first or second aspect, the modifying comprises inhibiting the phosphatidylethanolamine N-methyltransferase (PEMT) pathway in said cell.

In another embodiment of the first or second aspect, the inhibiting comprises reducing the quantity or activity of an enzyme, enzyme cofactor, precursor compound, or intermediate compound of said PEMT pathway.

In a further embodiment of the first or second aspect, the PEMT pathway enzyme is phosphatidylethanolamine methyltransferase.

In an additional embodiment of the first or second aspect, the PEMT pathway enzyme is phospholipid methyltransferase.

In one embodiment of the first or second aspect, the inhibiting comprises reducing the quantity or activity of transcription factor that regulates a PEMT pathway protein.

In a further embodiment of the first aspect, the transcription factor regulates inositol- 1- phosphate synthase expression.

In one embodiment of the first or second aspect, the inhibiting comprises reducing the quantity or activity of a protein in the cell, wherein said protein is selected from the group consisting of: inositol- 1 -phosphate synthase, CDP-diacylglycerol synthase (phosphatidate cytidylyltransferase), (e.g. CDP-diacylglycerol synthase 1, CDP-diacylglycerol synthase 2), casein kinase II, a beta regulatory subunit of casein kinase II, a protein homologous to Saccharomyces cerevisiae IN02/YDR123C, a protein homologous to Saccharomyces cerevisiae INO4/YOL108C, a protein homologous to Saccharomyces cerevisiae RTC2 YBR 147 W, a protein homologous to Saccharomyces cerevisiae MRPS35 Y GR 165 W, a protein homologous to Saccharomyces cerevisiae FLD1/YLR404W, and a protein homologous to Saccharomyces cerevisiae FLD1/YLR404W.

In another embodiment of the first or second aspect, the inhibiting comprises inhibiting the expression of a gene encoding a protein selected from the group consisting of: inositol- 1- ,

3 phosphate synthase, CDP-diacylglycerol synthase (phosphatidate cytidylyltransferase) (e.g. CDP-diacylglycerol synthase 1 , CDP-diacylglycerol synthase 2), casein kinase II, a beta regulatory subunit of casein kinase II, a protein homologous to Saccharomyces cerevisiae IN02/YDR123C, a protein homologous to Saccharomyces cerevisiae INO4 YOL108C, a protein homologous to Saccharomyces cerevisiae RTC2/YBR147W, a protein homologous to Saccharomyces cerevisiae MRPS35/YGR165W, a protein homologous to Saccharomyces cerevisiae FLD1/YLR404W, and a protein homologous to Saccharomyces cerevisiae FLD 1/YLR404W.

In one embodiment of the first or second aspect, the inhibiting comprises reducing the quantity or activity of a CDP-diacylglycerol synthase protein encoded by a gene comprising a sequence as defined in SEQ ID NO: 41.

In one embodiment of the first or second aspect, the inhibiting comprises reducing the quantity or activity of phosphatidylethanolamine methyltransferase and phospholipid methyltransferase.

In one embodiment of the first or second aspect, the inhibiting comprises reducing the quantity or activity of phosphatidylethanolamine methyltransferase, phospholipid methyltransferase, and a protein homologous to Saccharomyces cerevisiae FLD1 YLR404W.

In another embodiment of the first or second aspect, the inhibiting comprises reducing the quantity or activity of phosphatidylethanolamine methyltransferase, phospholipid methyltransferase and CDP-DAG synthase.

In one embodiment of the first or second aspect, the inhibiting comprises reducing the quantity or activity of phosphatidylethanolamine methyltransferase, phospholipid methyltransferase, and casein kinase 2.

In another embodiment of the first or second aspect, the inhibiting comprises reducing the quantity or activity of phosphatidylethanolamine methyltransferase, phospholipid methyltransferase, CDP-DAG synthase and casein kinase 2.

In a further embodiment of the first or second aspect, the cell is a bacterial cell, a yeast, an algal cell, or a plant cell.

In a further embodiment of the first or second aspect, the cell is a Chlamydomonas sp. microorganism.

In another embodiment of the first or second aspect, the method comprises inhibiting the expression of a gene in said cell. ,

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In one embodiment of the first or second aspect, the organism is a non-human organism.

In a third aspect, the invention provides a method for producing a genetically modified organism, the method comprising making at least one genetic modification to a cell of the organism that increases phosphatidic acid production in the cell, wherein triacylglycerol production in the cell is elevated compared to a corresponding wild-type cell, and said organism is a microorganism or plant.

In one embodiment of the third aspect, the genetic modification inhibits the phosphatidylethanolamine N-methyltransferase (PEMT) pathway in said cell.

In another embodiment of the third aspect, the genetic modification reduces the quantity or activity of an enzyme, enzyme cofactor, precursor compound, or intermediate compound of said PEMT pathway.

In a further embodiment of the third aspect, the PEMT pathway enzyme is phosphatidylethanolamine methyltransferase.

In an additional embodiment of the third aspect, the PEMT pathway enzyme is phospholipid methyltransferase

In one embodiment of the third aspect, the genetic modification reduces the quantity or activity of a transcription factor that regulates a PEMT pathway protein.

In a further embodiment of the third aspect, the transcription factor regulates inositol- 1- phosphate synthase expression.

In another embodiment of the third aspect, the genetic modification reduces the quantity or activity of a protein selected from the group consisting of: inositol- 1 -phosphate synthase, CDP-diacylglycerol synthase (phosphatidate cytidylyltransferase), (e.g. CDP- diacylglycerol synthase 1, CDP-diacylglycerol synthase 2), casein kinase II, a beta regulatory subunit of casein kinase II, a protein homologous to Saccharomyces cerevisiae IN02/YDR123C, a protein homologous to Saccharomyces cerevisiae INO4 YOL108C, a protein homologous to Saccharomyces cerevisiae RTC2/YBR147W, a protein homologous to. Saccharomyces cerevisiae MRPS35 YGR165W, a protein homologous to Saccharomyces cerevisiae FLD1/YLR404W, and a protein homologous to Saccharomyces cerevisiae FLD1 YLR404W.

In another embodiment of the third aspect, the genetic modification inhibits the expression of a gene in the organism encoding a protein selected from the group consisting of: inositol- 1 -phosphate synthase, CDP-diacylglycerol synthase (phosphatidate cytidylyltransferase) (e.g. CDP-diacylglycerol synthase 1, CDP-diacylglycerol synthase 2), .

5 casein kinase II, a beta regulatory subunit of casein kinase II, a protein homologous to Saccharomyces cerevisiae IN02/YDR123C, a protein homologous to Saccharomyces cerevisiae INO4/YOL108C, a protein homologous to Saccharomyces cerevisiae RTC2/YBR 147 W, a protein homologous to Saccharomyces cerevisiae MRPS35/YGR165W, a protein homologous to Saccharomyces cerevisiae FLD1/YLR404W, and a protein homologous to Saccharomyces cerevisiae FLD1/YLR404W.

In one embodiment of the third aspect, the genetic modification reduces the quantity or activity of a CDP-diacylglycerol synthase protein encoded by a gene comprising a sequence as defined in SEQ ID NO: 41.

In one embodiment of the third aspect, the genetic modification reduces the quantity or activity of phosphatidylethanolamine methyltransferase and phospholipid methyltransferase.

In another embodiment of the third aspect, the genetic modification reduces the quantity or activity of phosphatidylethanolamine methyltransferase, phospholipid methyltransferase and CDP-DAG synthase.

In one embodiment of the third aspect, the genetic modification reduces the quantity or activity of phosphatidylethanolamine methyltransferase, phospholipid methyltransferase, and casein kinase 2.

In another embodiment of the third aspect, the genetic modification reduces the quantity or activity of phosphatidylethanolamine methyltransferase, phospholipid methyltransferase, CDP-DAG synthase and casein kinase 2.

In a further embodiment of the third aspect, the genetically modified organism is a bacterium, plant or alga.

In a further embodiment of the third aspect, the genetically modified organism is a Chlamydomonas sp. organism. In a fourth aspect, the invention provides a method for producing a biofuel, the method comprising:

cultivating a genetically modified organism according to the second aspect, isolating triacylglycerols produced by the organism; and

transesterifying the triacylglycerols to produce the biofuel.

In one embodiment of the fourth aspect, the transesterifying is performed using a monohydric alcohol and a base catalyst.

In one embodiment of the fourth aspect, the biofuel is biodiesel.

In a fifth aspect, the invention provides use of a genetically modified organism produced by the method of the third aspect in a process for producing a biofuel. .

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In a sixth aspect, the invention provides a method for increasing triacylglycerol production in a cell, the method comprising reducing the quantity or activity of a synaptobrevin/vesicle-associated membrane protein (VAMP)-associated protein in the cell.

In one embodiment of the sixth aspect, the VAMP-associated protein is a protein homologous to Saccharomyces cerevisiae SCS2/YER120W.

In another embodiment of the sixth aspect, the VAMP-associated protein comprises a sequence defined in SEQ ID NO: 5, SEQ ID NO: 17, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO: 40.

In another embodiment of the sixth aspect, the quantity or activity of the VAMP- associated protein is reduced by inhibiting the expression of a gene encoding the VAMP in the cell.

In a further embodiment of the sixth aspect, the gene is a homologue of S. cerevisiae scs2.

In a further embodiment of the sixth aspect, the cell is a bacterium, yeast, plant or algal cell.

In a seventh aspect, the invention provides a method for producing a genetically modified organism, the method comprising making at least one genetic modification to a cell of the organism that reduces the quantity or activity of a synaptobrevin/vesicle-associated membrane protein (VAMP)-associated protein in the cell, wherein said organism is a microorganism or plant.

In one embodiment of the seventh aspect, the VAMP-associated protein is a protein homologous to Saccharomyces cerevisiae SCS2/YER120W.

In another embodiment of the seventh aspect, the VAMP-associated protein comprises a sequence defined in SEQ ID NO: 5, SEQ ID NO: 17, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO: 40.

In another embodiment of the seventh aspect, the genetic modification inhibits the expression of a gene encoding the VAMP-associated protein in the organism.

In a further embodiment of the seventh aspect, the genetically modified organism is a bacterium, yeast, plant or alga. In an eighth aspect, the invention provides a genetically modified organism produced in accordance with the method of the third aspect.

In a ninth aspect, the invention provides a genetically modified organism produced in accordance with the method of the seventh aspect.

In a tenth aspect, the invention provides use of a genetically modified organism produced by the method of the seventh aspect in a process for producing a biofuel. In an eleventh aspect, the invention provides a biofuel produced in accordance with the method of the fourth aspect, the use of the fifth aspect, or the use of the tenth aspect.

In one embodiment of the eleventh aspect, the biofuel is biodiesel.

In a further embodiment of the first, second, or sixth aspect, the cell is Chlamydomonas reinhardtii.

In a further embodiment of the first, second, or sixth aspect, the cell is a Saccharomyces sp. microorganism.

In a further embodiment of the first, second, or sixth aspect, the cell is Saccharomyces cerevisiae.

In a further embodiment of the first, second, or sixth aspect, the cell is a plant cell.

In a further embodiment of the first, second, or sixth aspect, the cell is a plant cell selected from an Arabidposis sp. plant cell, a Glycine sp. plant cell, a Brassica sp. plant cell, and a Ricinus sp. plant cell.

In a further embodiment of the first, second, or sixth aspect, the cell is a plant cell selected from a Brassica rapa plant cell and an Arabidposis thaliana plant cell.

In a further embodiment of the third, seventh or eighth aspect, the genetically modified organism is Chlamydomonas reinhardtii.

In a further embodiment of the third, seventh or eighth aspect, the genetically modified organism is a Saccharomyces sp. microorganism.

In a further embodiment of the third, seventh or eighth aspect, the genetically modified organism is Saccharomyces cerevisiae.

In a further embodiment of the third, seventh or eighth aspect, the genetically modified organism is a plant.

In a further embodiment of the third, seventh or eighth aspect, the genetically modified organism is a plant selected from an Arabidposis sp. plant, a Glycine sp. plant, a Brassica sp. plant, and a Ricinus sp. plant.

In a further embodiment of the third, seventh or eighth aspect, the genetically modified organism is a plant selected from a Brassica rapa plant and an Arabidposis thaliana plant.

In another embodiment of the first, second, third, fourth, fifth or eighth aspect, the CDP-diacylglycerol synthase (phosphatidate cytidylyltransferase) protein comprises a sequence as set forth in any one of SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NOs 18-21, a variant of any one of said sequences, or a fragment of any one of said sequences.

In another embodiment of the first, second, third, fourth, fifth or eighth aspect, the casein kinase II protein comprises a sequence as set forth in any one of SEQ ID Nos 2-3, SEQ ID NOs 8-9, SEQ ID Nos 22-27, a variant of any one of said sequences, or a fragment of any one of said sequences.

In another embodiment of the first, second, third, fourth, fifth or eighth aspect, the protein homologous to Saccharomyces cerevisiae RTC2/YBR147W is a transmembrane family protein. The transmembrane family protein may be a PQ-loop repeat family protein.

In another embodiment of the first, second, third, fourth, fifth or eighth aspect, the protein homologous to Saccharomyces cerevisiae RTC2/YBR147W comprises a sequence as set forth in any one of SEQ ID NO: 4, SEQ ID NOs 13-16, SEQ ID NOs 29-36, a variant of any one of said sequences, or a fragment of any one of said sequences.

In another embodiment of the first, second, third, fourth, fifth or eighth aspect, the protein homologous to Saccharomyces cerevisiae IN02 YDR123C comprises a sequence as set forth in any one of SEQ ID NO: 10, a variant of SEQ ID NO: 10, or a fragment of SEQ ID NO: 10.

In another embodiment of the first, second, third, fourth, fifth or eighth aspect, the protein homologous to Saccharomyces cerevisiae MRPS35 YGR165W comprises a sequence as set forth in any one of SEQ ID NO: 11 , a variant of SEQ ID NO: 11 , or a fragment of SEQ ID NO: 11.

In another embodiment of the first, second, third, fourth, fifth or eighth aspect, the inhibiting comprises reducing the quantity or activity of a phospholipid N-methyltransferase protein in the cell.

In another embodiment of the first, second, third, fourth, fifth or eighth aspect, the phospholipid N-methyltransferase protein comprises a sequence as set forth in any one of SEQ ID NO: 12, SEQ ID NO: 28, a variant of any one of said sequences, or a fragment of any one of said sequences.

In another embodiment of the first, second, third, fourth, fifth or eighth aspect, the gene encoding a CDP-diacylglycerol synthase (phosphatidate cytidylyltransferase) protein comprises a sequence as set forth in any one of SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 54, a variant of any one of said sequences, or a fragment of any one of said sequences,

In another embodiment of the first, second, third, fourth, fifth or eighth aspect, the gene encoding a casein kinase II protein comprises a sequence as set forth in any one of SEQ ID NO: 47, SEQ ID NO: 51, a variant of any one of said sequences, or a fragment of any one of said sequences, ,

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In another embodiment of the first, second, third, fourth, fifth or eighth aspect, the gene encoding a protein homologous to Saccharomyces cerevisiae RTC2/YBR147W is a transmembrane family protein. The transmembrane family protein may be a PQ-loop repeat family protein.

In another embodiment of the first, second, third, fourth, fifth or eighth aspect, the gene encoding a protein homologous to Saccharomyces cerevisiae RTC2/YBR147W comprises a sequence as set forth in any one of SEQ ID NO: 46, SEQ ID NO: 52, a variant of any one of said sequences, or a fragment of any one of said sequences,

In another embodiment of the first, second, third, fourth, fifth or eighth aspect, the inhibiting comprises inhibiting the experession of a gene encoding a phospholipid N- methyltransferase protein in the cell.

In another embodiment of the sixth, seventh, ninth, or tenth aspect, the VAMP- associated protein or protein homologous to Saccharomyces cerevisiae SCS2/YER120W comprises a sequence as set forth in any one of SEQ ID NO: 5, SEQ ID NO: 17, SEQ ID NOs 37-40, a variant of any one of said sequences, or a fragment of any one of said sequences.

In another embodiment of the sixth, seventh, ninth, or tenth aspect, the gene encoding the VAMP-associated protein or protein homologous to Saccharomyces cerevisiae SCS2 YER120W comprises a sequence as set forth in any one of SEQ ID NO: 48, SEQ ID NO: 53, a variant of any one of said sequences, or a fragment of any one of said sequences.

In another embodiment of the first, second, fourth, fifth, sixth, eighth, or eleventh aspect, the triacylglycerol comprises at least one saturated fatty acid chain.

In an additional embodiment of the first, second, fourth, fifth, sixth, eighth, or eleventh aspect, the triacylglycerol comprises at least one short chain fatty acid comprising less than 14 carbon atoms.

In one embodiment of any one of the first to tenth aspects, the variant has a level of sequence identity with said sequence selected from the group consisting of at least 98% sequence identity, at least 95% sequence identity, at least 90% sequence identity, at least 85% sequence identity, at least 80% sequence identity, and at least 75% sequence identity,

In one embodiment of any one of the first to tenth aspects, the variant is a homologous gene aor a homologous protein in an organism of a different species, genus, fanily or class.

In one embodiment of any one of the first to tenth aspects, the fragment is less than 1000 base pairs in length, less than 900 base pairs in length, less than 800 base pairs in length, less than 700 base pairs in length, less than 600 base pairs in length, less than 500 ,

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base pairs in length, less than 400 base pairs in length, less than 300 base pairs in length, less than 200 base pairs in length, less than 100 base pairs in length, less than 75 base pairs in length, less than 50 base pairs in length, less than 40 base pairs in length, less than 30 base pairs in length, less than 20 base pairs in length, or less than 10 base pairs in length,

In one embodiment of any one of the first to tenth aspects, the fragment is less than 1000 amino acid residues in length, less than 900 amino acid residues in length, less than 800 amino acid residues in length, less than 700 amino acid residues in length, less than 600 amino acid residues in length, less than 500 amino acid residues in length, less than 400 amino acid residues in length, less than 300 amino acid residues in length, less than 200 amino acid residues in length, less than 100 amino acid residues in length, less than 75 amino acid residues in length, less than 50 amino acid residues in length, less than 40 amino acid residues in length, less than 30 amino acid residues in length, less than 20 amino acid residues in length, or less than 10 amino acid residues in length.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the invention will now be described, by way of example only, with reference to the accompanying figures wherein:

Figure 1 provides microscopy images and a graph showing yeast fldlA and nine additional mutant strains produce "supersized" LDs (SLDs). Cells were grown in YPD or SC medium until early stationary phase, stained with Nile red, and examined by fluorescence microscopy. Bar, 5 urn. (A) LDs of WT and fldlA. (B) LDs of mutant strains defective in either CDP-choline pathway (ckiJA, pctlA, and cptlA) or PEMT pathway (cho2A, opi3A, ino2A, and ino4A) of PC synthesis. (C) Supersized LDs observed in rtc2A, mrps35A, ckblA and ckb2A. (D) LDs of a yeast strain with the CDS1 gene under the control of a tetracycline- regulated promoter grown in the presence or absence of doxycycline. (E) Transmission electron microscopic examination of LDs in WT, fldlA, cho2A, opi3A, ino2A, ino4A, and cdsl strains cultured in SC medium. Bar, 1 μπι. (F) Relative cellular amounts of TAG and SE. #, /7<0.05; *,ρ<0.01, compared to WT.

Figure 2 provides microscopy images and a graph showing treatment of different phospholipid precursors exerts distinct effects on the formation of SLDs. WT and mutants were cultured in SC media supplemented without (con) or with 1 mM choline (C), 1 mM ethanolamine (E), or 75 μΜ inositol (I) to stationary phase and observed under a fluorescence microscope. (A) Microscopic images. Bar, 5 μπι. (B) Percentage of cells containing SLDs. *, /K0.01. ,

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Figure 3 provides graphs illustrative of a link between the formation of SLDs and an elevated level of cellular PA. (A) and (B) Quantitation of cellular PA in WT and mutants. Cells were grown in SC medium to early stationary phase, harvested, and lyophilized. Lipids were extracted and PA levels were determined by LC-MS. *, / 0.05, compared to WT. (C) Overexpression of PAH1 and DPP1 significantly reduces the formation of SLDs in fldlA, inp2A, and ino4A strains. Cells transformed with BG1805-PAH1, BG1805-DPP1 (both from Open Biosystems) or empty vector were cultured in synthetic galactose medium (2% galactose, 0.67% yeast nitrogen base, and amino acids) to stationary phase, stained with Nile red, and examined for the presence of SLDs.

Figure 4 shows graphs illustrating Fldlp and cellular PA expression. (A) The gene expression fold changes (fldlAfWT) for selective genes involved in phospholipid metabolism as determined by microarray analysis. Cells were cultured in YPD medium until log phase (OD6oo~0.8). The expression levels of INOl and OPIS were significantly upregulated in fldlA cells. *, /XO.01 (ANOVA, FDR <0.05). (B) Relative mRNA levels of INOl and OPI3 as determined by qPCR in WT and fldlA strains and normalized to ACT1. *, / θ.01. (C) WT and fldlA cells without or with (+Ino) inositol treatment were grown to late log phase. Microsomes were isolated as described in methods. Lipids were extracted and the amounts of PA were determined by mass-spectrometry. #, /?<0.05, compared to WT.

Figure 5 provides a microscopy image and graphs illustrating SLD formation in yeast cells deficient in PA phosphatase activity. (A) and (B) Formation of SLDs in pahlA, but not in dgalA or dgalA IrolA strains. Bar, 5 μπι. (C) Relative cellular TAG amounts quantified by thin layer chromatography and densitometry. (D) Addition of 1 mM membrane-permeable DAG analog (1,2-dioctanoyl-sn-glycerol) and 1 mM oleate, but not oleate alone, significantly elevated the percentage of pahlA cells accumulating supersized LDs. Cells with supersized LDs were counted and the percentages were presented as mean±SD.

Figure 6 provides graphs showing (A) PE/PL ratio of LDs isolated from WT and mutants grown in SC medium, and of LDs isolated from ino4A,fldlA, and cdsl cells cultured in SC medium without (con) or with the addition of choline (C), ethanolamine (E), or inositol (I); (B) PL/TAG ratio of LDs isolated from WT and mutants grown in SC medium or YPD medium, and of LDs isolated from ino4A, fldlA, and cdsl cells cultured in SC medium without (con) or with the addition of choline (C), ethanolamine (E), or inositol (I). *, /?<0.01 ; **, / 0.05, compared to WT.

Figure 7 provides graphs showing rtc2A and mrps35A strains exhibit a higher PE/PC ratio. WT and mutants were cultured in SC medium to early stationary phase. Cellular .

12 amounts of major phospholipid species were analyzed by LC-MS. #, p<0.05; *, p<0.0 , compared to WT.

Figure 8 provides microscopy images showing that mutant strains that produce SLDs display enhanced LD fusion activity in vivo. Cells were grown in SC media until mid-log phase (OD6oo~1.5), stained with Nile red and examined for LD fusion activities under fluorescence microscope. Cells in which two or several LDs lay close together were targeted. Images were taken at a 0.5 s interval. Bar, 5 μπι.

Figure 9 provides a graph indicative of the stability and size of TAG containing artificial droplets consisting of the indicated molar ratios of the phospholipids were determined by light scattering. The artificial droplets were generated by sonication, and liposomes formed at the same time were removed by density gradient centrifugation. The stability/number of LDs was measured by light scattering. Values are mean ± SD of three experiments.

Figure 10 is a diagram showing biosynthetic pathways of major phospholipids and TAG in S. cerevisiae. PA, phosphatide acid; CDP-DAG, CDP-diacylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; PE, phosphatidylethanolamine; PMME, phosphatidylmonomethylethanolamine; PDME, phosphatidyldimethylethanolamine; PC, phosphatidylcholine; DAG, diacylglycerol; TAG, triacylglycerol. Grey-shaded enzymes are under the control of the Ino2p-Ino4p complex

Figure 11 provides microscopy images and a timecourse graph showing in vivo TAG mobilization of WT,fldlA, ino4A, and cdsl in the presence of 10 μg/ml cerulenin. Cells were grown in SC medium for 24 hr, and refreshed in YPD medium supplemented with 10 μg ml cerulenin to OD600~1.5. Cells were collected at indicated time points, followed by fluorescence microscopy and lipid analysis.

Figure 12 provides graphs indicating the effect of DPP1 and PAH1 expression on cellular lipids. (A) Total cellular PA as measured by mass-spectrometry. *, p<0.0\, compared to vector control. (B) Major phospholipids on lipid droplets measured by mass-spectrometry.

Figure 13 shows relative cellular levels of PC, PE and PI in WT and mutants as determined by thin layer chromatography (TLC). Densitometric analysis was performed using the Image Gauge 4.0 software (Fujifilm Science Lab).

Figure 14 provides fluorescent microscopy images showing lipid droplet accumulation in the algae Chlamydomonas. (A) wild type (B) transformed with RNAi construct. .

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DEFINITIONS

As used in this application, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a plant cell" also includes a plurality of plant cells.

As used herein, the term "comprising" means "including." Variations of the word "comprising", such as "comprise" and "comprises," have correspondingly varied meanings. Thus, for example, a polynucleotide "comprising" a sequence of nucleotides may consist exclusively of that sequence, or, may include one or more additional sequences of nucleotides.

As used herein, the term "biofuel" includes any energy-containing fuel product derived from the processing of a lipid. Non-limiting examples of biofuels include oil products (i.e. bio-oils), biodiesel, and alcohols (e.g. ethanol and butanol).

Any description of prior art documents herein, or statements herein derived from or based on those documents, is not an admission that the documents or derived statements are part of the common general knowledge of the relevant art.

For the purposes of description all documents referred to herein are incorporated by reference in their entirety unless otherwise stated.

DETAILED DESCRIPTION

A number of current methods for biofuel production utilise lipids generated by plants and/or microorganisms (e.g. algae and bacteria). The economic feasibility of current methods depends largely on the yield and/or characteristics of lipids harvested from the plants and/or microorganisms utilised.

The present inventors have identified that the quantity and/or quality of lipids in plants and microorganisms can be improved by raising intracellular levels of phosphatidic acid. In particular, increasing phosphatidic acid has been identified to cause increased production of glycerolipids (e.g. triacylglycerols and sterol esters) including those comprising short and/or saturated acyl chains. The present inventors have also identified that increased cellular phosphatidic acid is strongly associated with the formation of large/supersized lipid droplets within a cell. Without limitation to a particular mode of action, it is postulated that increasing cellular levels of phosphatidic acid serves to enhance the fusion properties of intracellular lipid droplets resulting in the formation of large/supersized lipid droplets within cells. These large/supersized lipid droplets contain large numbers of certain lipids (e.g. glycerolipids including triacylglycerols) with characteristics advantageous for biofuel production (e.g. .

14 short and/or saturated acyl chains). Further, large/supersized lipid droplets provide a much more efficient way for the cell to store energy in comparison to having a large number of small lipid droplets.

Accordingly, the invention provides methods for increasing the production of lipids in a plant or microorganism, the methods comprising increasing cellular levels of phosphatidic acid. Methods for increasing the energy-storage potential of a cell in a plant or microorganism are also provided, the methods comprising increasing cellular levels of phosphatidic acid.

The generation of plants and microorganisms with improved lipid-producing qualities is desirable to increase the efficiency of biofuel production and enhance its utility as a replacement for fossil fuels. To this end, the invention provides genetically modified plants and microorganisms in which the quantity and/or quality of lipids (e.g. triacylglycerols and sterol esters) is increased compared to corresponding wild-type microorganisms or plants. Genetically modified plants and microorganisms of the invention generally comprise at least one genetic alteration that increases cellular levels of phosphatidic acid.

Additional aspects of the invention relate to methods for generating biofuels (e.g. biodiesel) utilising lipids produced by genetically modified plants and/or microorganisms described herein.

Microorganisms and plants

The invention provides methods for increasing the production of lipids in a cell. In some embodiments, the lipids are glycerolipids (e.g. triacylglycerols and sterol esters). In some embodiments, the glycerolipids comprise short and/or saturated acyl chains.

The cell may be any cell capable of lipid production. For example, the cell may be a lipid-producing unicellular organism (e.g. a bacterium, microalga or fungus). Alternatively, the cell may be derived from or exist as a constituent of a lipid-producing multicellular organism (e.g. a plant).

Accordingly, the methods of the invention may be used to increase lipid production in a plant cell. Non-limiting examples of plant cells in which lipid production may be increased include Azadirachta indica (neem), Brassica sp. (mustard), Carapa sp. (andiroba), Orbignia sp (babassu), Hordeum vulgare (barley), Ricinus communis (castor), Camelina sativa (camelina), Brassica campestris (canola/rapeseed), Cocos nucifera (coconut), Zea mays (corn), Dipteryx odorata (cumaru), Cynara cardunculus (artichoke thistle), Arachis hypogaea (groundnut), Pongamia glabra (karanja), Jatropha curcas (jatropha), Lauyrus sp. (bay laurel), .

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Lesquerella fendleri (lesquerella), Caryocar sp. (piqui), Elaeis sp. (palm), Sesamum indicum (sesame), Sorghum sp. (grasses) and Glycine max (soybean) species.

The methods may be used to increase lipid production in a bacterial cell. Non-limiting examples of bacteria in which lipid production may be increased include Rhodococcus sp. (e.g. R. opacus, R. fascians, R. erythroplois, R. glutinis, Nocardia sp.), (e.g. N. corallina, N. asteroids, N. globerula, N. restricta), Gordonia sp. (e.g. Gordonia amarae), Pseudomonas sp. (e.g. P. aeruginosa), Acinetobacter sp. (e.g. A. lwoffii) and cyanobacteria.

The methods may be used to increase lipid production in a fungal cell. Non-limiting examples of fungi in which lipid production may be increased include Candida sp. (e.g. C. valida, C. utilus), Yarrowia sp. (e.g. Y. lypolytica), Rhodotorula (e.g. R. gracilis) and Mortiorella sp. (e.g. M. peryronel).

The methods may be used to increase lipid production in an algal cell. Preferably, the algal cell is a microalgal cell. Non-limiting examples of algae in which lipid production may be increased include Chlorophytes (e.g. Ankistrodesmus sp., Botryococcus sp., Chlamydomonas sp. (including C. reinhardtii), Chlorella sp., Chlorococcum sp., Dunaliella sp., onoraphidium sp., Oocystis sp., Scenedesmus sp., and Tetraselmis sp.), cyanophytes (e.g. Oscillatoria sp. and Synechococcus sp.), chrysophytes (e.g. Boekelovia sp.), haptophytes (e.g. Isochrysis sp. and Pleurochysis sp.), and bacillariophytes (e.g. Amphipleura sp., Amphora sp., Chaetoceros sp., Cyclotella sp., Cymbella sp., Fragilaria sp., Hantzschia sp., Navicula sp., Nitzschia sp., Phaeodactylum sp., and Thalassiosira sp.).

Lipid production

As contemplated herein, "increasing lipid production" in a cell refers to a process whereby the production of at least one type of lipid in the cell is increased compared to the production of that same lipid in the cell prior to performing the method (under the same biological conditions). It will be understood that "increasing lipid production" in a cell does not necessarily exclude decreasing the production of particular type(s) of lipid(s) in the cell, provided that the production of at least one type of lipid is increased in the cell.

Methods of the invention may be utilised to increase the production of any lipid. For example, the methods may be utilised to increase the production of any one or more of fatty acyls, glycerolipids, glycerophospholipids, cardiolipins, sphingolipids, prenols or sterols in a cell. In some embodiments, the methods may be used to increase the production of glycerolipids in the cell. The glycerolipids may be, for example, triacyclglycerols and/or sterol esters.

The increased production of lipids in the cell may be detected using methods known in the art. Exemplary techniques include reverse-phase high performance liquid chromatography and mass spectrometry. Total lipid content in a cell may be determined, for example, using the technique of Bligh and Dyer, (see Bligh and Dyer, (1959), "A rapid method of total lipid extraction and purification", Canadian Journal of Biochemistry and Physiology, 37(8):911- 917). An exemplary method for the quantification of triacylglycerols in cellular lipid extracts is provided in Van Veldhoven et al., (1997), "Lipase-based quantitation of triacylglycerols in cellular lipid extracts: Requirement for presence of detergent and prior separation by thin- layer chromatography", Lipids, 32(12): 1297-1300. Additional non-limiting examples of methods for quantifying lipids are provided in United States Patent No. 4370311 (issued on 25 January, 1983 to Ilekis, John V) and United States Patent No. 5491093 (issued on 13 February, 1996 to Yamamoto, et al.).

Methods of the invention may be used to increase the production of triacylglycerols (TAGs) in a cell. A "triacylglycerol" (also known as a "triglyceride") as contemplated herein is a glycerolipid formed from the binding of three fatty acids to a glycerol molecule. The three fatty acids may be identical. Alternatively, two of the fatty acids may be identical. Alternatively, each fatty acid of the triacylglycerol may be a different fatty acid.

The triacylglycerol may comprise fatty acids of any length. In certain embodiments of the invention, the triacylglycerol comprises at least one long chain fatty acid having greater than 18 carbon atoms. In other embodiments of the invention, the triacylglycerol comprises at least one medium chain fatty acid having 14 carbon atoms to 18 carbon atoms. In preferred embodiments the triacylglycerol comprises at least one short chain fatty acid having less than 14 carbon atoms.

The triacylglycerol may comprise saturated and/or unsaturated fatty acids. The unsaturated fatty acid may comprise any number of double and/or triple carbon bonds. It will be understood that triacylglycerols comprising one or more unsaturated fatty acids as contemplated herein include cis and trans configurational isomers of those unsaturated fatty acid(s). The triacylglycerol may comprise an unsaturated fatty acid of any length. For example, the unsaturated fatty acid may be a short chain fatty acid, a medium chain fatty acid (e.g. 9-hexadecenoic acid (16:1); 9-octadecenoic acid (18:1), 12-hydroxy-9-octadecenoic acid (18: 1), 11 octadecenoic acid (18:1), 9,12-octadecadienoic acid (18:2), 9,12,15- octadecatrienoic acid (18:3), 6,9,12-octadecatrienoic acid (18:3)), or a long chain fatty acid (e.g. 9-eicosenoic acid (20:1), 5,8,11,14-eicosatetraenoic acid (20:4), 5,8,11,14,17- eicosapentaenoic acid (20:5), 13-docosenoic acid ( 22:1), 4,7,10,13,16,19-docosahexaenoic acid (22:6)).

The triacylglycerol may comprise a saturated fatty acid of any length. For example, the saturated fatty acid may be a short chain fatty acid (e.g. butanoic acid (C4:0); hexanoic acid (C6:0); octanoic acid (C8:0), decanoic acid (C10:0), dodecanoic acid (C12:0)), a medium chain fatty acid (e.g. tetradecanoic acid (C14:0); hexadecanoic acid (C16:0); octadenoic acid (CI 8:0)), or a long chain fatty acid (e.g. eicosanoic acid (20C); docosanoic acid (22C); tetracosanoic acid (24C)).

The production of biodiesel from triacylglycerols with shorter acyl chains may reduce the level of viscosity. Accordingly, in certain embodiments a triacylglycerol produced in accordance with the methods of the invention comprises at least one saturated medium chain fatty acid having 16 carbon atoms and/or at least one saturated medium chain fatty acid having 14 carbon atoms. More preferably, a triacylglycerol produced in accordance with the methods of the invention comprises at least one saturated short chain fatty acid having 12 carbon atoms, more preferably 10 carbon atoms, and still more preferably less than 10 carbon atoms.

Phosphatide acid production and the PEMT pathway

Increasing cellular levels of phosphatidic acid (1,2-Diacyl-sn-glycerol 3 -phosphate) is demonstrated herein to correlate with increased production of glycerolipids (e.g. triacylglycerols and/or sterol esters), and the production of glycerolipids with short and/or saturated fatty acid chains.

Accordingly, in certain aspects the invention provides methods for increasing the production of a lipid in a cell by raising phosphatidic acid production in the cell.

Phosphatidic acid production may be increased in the cell by any means.

For example, phosphatidic acid production may be increased in a cell by increasing the quantity and/or activity of one or more proteins or compounds of a lipid biosynthetic pathway of the cell. Alternatively, phosphatidic acid production may be increased in a cell by decreasing the quantity or activity of one or more proteins in a lipid biosynthetic pathway of the cell.

Non-limiting examples of such proteins include enzymes, transcription factors, regulatory proteins, intermediate proteins, and precursor proteins. ,

18

The quantity and/or activity of the protein may be increased or decreased by any means including, but not limited to, modifying the expression of a gene encoding the protein, modifying post-translational processing of the protein, agonizing a biological function of the protein, or antagonizing a biological function of the protein.

In some embodiments, phosphatidic acid production in a cell may be increased by increasing the quantity and/or activity of one or more proteins or compounds involved in a lipid biosynthetic pathway. For example, the quantity and/or activity of a protein (e.g. a precursor protein, an intermediate protein, an enzyme, a regulatory protein and/or a transcription factor) or compound in a pathway that positively regulates phosphatidic acid synthesis, either directly or indirectly, may be increased for the purpose of raising phosphatidic acid production in the cell. Non-limiting examples of proteins or compounds in this category include glycerol, glycerol-3 -phosphate, glycerol kinase (see EC 2.7.1.30 of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology), dihydroxyacetonephosphate, glycerol-3 -dehydrogenase (EC 1.1.99.5, EC 1.1.1.94, EC 1.1.1.8), acetyl-CoA precursors, 1-Acyl-sn-glycerol 3-phosphate, Glycerol-3-phosphate O-acyltransferase (EC 2.3. L15), and l-Acylglycerol-3 -phosphate O-acyltransferase (EC 2.3.1.51). The quantity and/or activity of such proteins may be increased by increasing the expression of genes encoding them.

In other embodiments, phosphatidic acid production in a cell may be increased by reducing the quantity and/or activity of one or more proteins or compounds involved in a lipid biosynthetic pathway. For example, the quantity and/or activity of a protein (e.g. a precursor protein, an intermediate protein, an enzyme, a regulatory protein and/or a transcription factor) or compound in a pathway that negatively regulates phosphatidic acid synthesis, either directly or indirectly, may be increased for the purpose of raising phosphatidic acid production in the cell. The quantity and/or activity of such proteins may be decreased by reducing the expression of genes encoding them.

In certain embodiments, phosphatidic acid levels may be increased in the cell by inhibiting the phosphatidylemanolamine N-methyltransferase (PEMT) pathway. As is known to the skilled addressee, the PEMT pathway involves the methylation of phosphatidylethanolamine to phosphatidylcholine in three steps by two methyltransferases, phosphatidylethanolamine N-methyltransferase (EC 2.1.1.17) and phospholipid methyltransferase (EC 2.1.1.16).

In accordance with the methods of the invention, "inhibiting" the PEMT pathway in a cell encompasses any means of reducing the occurrence or rate of one or more steps in the .

19

PEMT pathway. This may be achieved by targeting one or more components of the PEMT pathway directly, for example by reducing the quantity or activity of an enzyme, enzyme cofactor, precursor compound, and/or intermediate compound in the pathway. Additionally or alternatively, "inhibiting" the PEMT pathway in a cell may be achieved indirectly, for example, by increasing or decreasing the quantity and/or activity of other biological molecules that are not components within the PEMT pathway but nonetheless exert an influence on the pathway. For example, the activity of regulatory proteins (e.g. transcription factors) having effect on the expression of PEMT pathway proteins may be modified. Additionally or alternatively, the quantity and/or activity of component(s) in other interrelated lipid biosynthetic pathways (or the operation of these pathways in general) may be altered thereby inhibiting the PEMT pathway.

The skilled addressee can readily determine whether the PEMT pathway is inhibited in a given cell or microorganism by measuring PEMT pathway activity in that cell, and comparing it to PEMT pathway activity measured in substantially similar or identical untreated/wild-type cells (i.e. control cells). Assays for measuring PEMT pathway activity are well known in the art (e.g. radio-enzymatic assays). Non-limiting examples of suitable assays include those described in Hirata and Axelrod, (1978), "Enzymatic synthesis and rapid translocation of phosphatidylcholine by two methyltransferases in erythrocyte membranes" Proc. Natl. Acad. Sci. U.S.A.75, 2348-2352; Hirata et al, (1978), "Identification and properties of two methyltransferases in conversion of phosphatidylethanolamine to phosphatidylcholine", Proc.Natl. Acad. Sci. U.S.A. 75, 1718-1721; Ridgway and Vance, (1992), "Phosphatidylethanolamine N-methyltransferase from rat liver", Methods Enzymol. 209, 366-374; and Duce et al., (1988), "S-Adenosyl- L-methionine synthetase and phospholipid methyltransferase are inhibited in human cirrhosis", Hepatology 8: 65-68.

In some embodiments of the invention, the PEMT pathway is inhibited by reducing the quantity or activity of:

0) phospholipid methyltransferase (EC 2.1.1.16);

(ϋ) phosphatidylethanolamine methyltransferase (EC 2.1.1.17);

(iii) inositol- 1 -phosphate synthase (EC 5.5.1.4);

(iv) CDP-diacylglycerol synthase (EC: 2.7.7.41);

(v) casein kinase II (EC 2.7.11.1);

(vi) a beta regulatory subunit of casein kinase II (EC 2.7.11.1);

(vii) a transcription factor that positively regulates one or more components of the PEMT pathway - for example, Saccharomyces cerevisiae IN02/YDR123C and/or ,

20

INO4/YOL108C, or a protein homologous to Saccharomyces cerevisiae IN02/YDR123C or INO4/YOL108C;

(viii) Saccharomyces cerevisiae RTC2 YBR147W, or a protein homologousto Saccharomyces cerevisiae RTC2/YBR147W;

(ix) Saccharomyces cerevisiae MRPS35/Y GR165W or a protein homologous to Saccharomyces cerevisiae MRPS35 YGR165W;

(x) Saccharomyces cerevisiae FLD1/YLR404W, or a protein homologous to Saccharomyces cerevisiae FLD1/YLR404W;

(xi) any combination of two or more of (i)-(x).

The skilled addressee will understand that "homologous proteins" in this context are identical or similar proteins from a different microorganism or plant sharing substantially the same biological function or activity as the original protein referred to.

In some embodiments, the PEMT pathway is inhibited by reducing the quantity or activity of: a Brassica sp. plant CDP-diacylglycerol synthase protein comprising the sequence defined by SEQ ID NO:l; a Ricinus sp. plant CDP-diacylglycerol synthase protein comprising the sequence defined by SEQ ID NO:6; a Glycine sp. plant CDP-diacylglycerol synthase protein comprising the sequence defined by any one of SEQ ID NO: 18, SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21 ; or a Chlamydomonas sp. protein encoded by the gene C 30067, the gene comprising the sequence set forth in SEQ ID NO: 41.

In some embodiments, the PEMT pathway is inhibited by reducing the quantity or activity of: a Brassica sp. plant casein kinase II protein comprising the sequence defined by SEQ ID NO: 2 or SEQ ID NO: 3; a Ricinus sp. plant casein kinase II protein comprising the sequence defined by SEQ ID NO: 8 or SEQ ID NO: 9; or a Glycine sp. plant casein kinase II protein comprising the sequence defined by any one of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27.

In some embodiments, the PEMT pathway is inhibited by reducing the quantity or activity of: a Brassica sp. plant protein comprising the sequence defined by SEQ ID NO:4; a Ricinus sp. protein comprising the sequence defined by any one or more of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16; or a Glycine sp. protein comprising the sequence defined by any one or more of SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36.

In some embodiments, the PEMT pathway is inhibited by reducing the quantity or activity of a Ricinus sp. phosphatidylethanolamine methyltransferase protein comprising the sequence defined by SEQ ID NO: 7. ,

21

In some embodiments, the PEMT pathway is inhibited by reducing the quantity or activity of: a Ricinus sp. phospholipid methyltransferase protein comprising the sequence defined by SEQ ID NO: 12; or a Glycine sp. phospholipid methyltransferase protein comprising the sequence defined by SEQ ID NO: 28.

In some embodiments, the PEMT pathway is inhibited by reducing the quantity or activity of a transcription factor that positively regulates one or more components of the PEMT pathway from Ricinus sp., comprising the sequence defined by SEQ ID NO: 10.

In some embodiments, the PEMT pathway is inhibited by reducing the quantity or activity of a Ricinus sp. protein comprising the sequence defined by SEQ ID NO: 11.

It will be understood that "reducing" the quantity or activity of a protein as contemplated herein encompasses any reduction of the quantity or activity of the protein when compared to its normal/standard quantity or activity under any given condition including, but not limited to, complete loss of function.

In some embodiments of the invention, the PEMT pathway is inhibited by inhibiting the expression of a gene encoding a protein in a lipid biosynthetic pathway of the cell (e.g. an enzyme, transcription factor, regulatory protein, intermediate protein, or precursor protein). For example, the PEMT pathway may be inhibited by inhibiting the expression of a gene encoding any one of the proteins referred to under (i) - (xi) above, and/or a gene encoding a protein comprising a sequence defined by any one or more of SEQ ID NOs 1 -41. It will be understood that "inhibiting" gene expression as contemplated herein encompasses any reduction of gene expression when compared to the normal/standard expression of the gene under any given condition, including, but not limited to, complete loss of gene expression.

VAMP-associated proteins

In additional embodiments of the invention, the production of glycerolipids is increased in a cell or microorganism by reducing the quantity or activity of a vesicle-associated membrane protein-associated protein (VAMP-associated protein).

The VAMP-associated protein may be Saccharomyces cerevisiae SCS2/YER120W or a homologous protein of another microorganism or plant.

The VAMP-associated protein may comprise a sequence defined in SEQ ID NO: 5, SEQ ID NO: 17, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO: 40.

The quantity or activity of the VAMP-associated protein may be reduced by inhibiting the expression of a gene encoding the VAMP. The gene may be a homologue of S. cerevisiae scs2. Combinations

It will be understood that the methods of inhibiting the phosphatidylethanolamine N- methyltransferase (PEMT) pathway may be achieved by:

(a) reducing the quantity or activity of any combination of two or more of the proteins set out under (i)-(x) above;

(b) reducing the quantity or activity of any combination of two or more proteins comprising a sequence defined by any one of SEQ ID NOs: 1-4, 6-16, or 18-36;

(c) reducing the quantity or activity of any combination of: at least one protein set out under (i)-(x) above and at least one protein comprising a sequence defined by any one of SEQ ID NOs: 1-4, 6-16, or 18-36;

(d) reducing the quantity or activity of any combination of: at least one protein set out under (i)-(x) above and at least one vesicle-associated membrane protein (VAMP)-associated protein including, but not limited to, at least one VAMP comprising any one of SEQ ID NO: 5, SEQ ID NO: 17, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO: 40;

(e) reducing the quantity or activity of any combination of: at least one protein set out under (i)-(x) above, and at least one protein comprising a sequence defined by any one of SEQ ID NOs: : 1-4, 6-16, or 18-36, and at least one vesicle-associated membrane protein (VAMP)-associated protein including, but not limited to, a VAMP-associated protein comprising any one of SEQ ID NO: 5, SEQ ID NO: 17, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO: 40; SEQ ID Nos 19-21;

(f) reducing the quantity or activity of a protein homologous to Saccharomyces cerevisiae FLD1 YLR404W in combination with any one of (a) - (e) above.

(g) performing any one or more of (a) - (f) above by altering the expression of genes encoding the protein(s).

Inhibition of gene expression

Inhibiting the expression of a gene in a cell in accordance with the methods of the invention can be performed using methods known in the art.

For example, the expression of a gene may be inhibited by reducing or eliminating transcription. Levels of gene transcription can be measured by any technique known in the art, including, for example, by transcription quantitative polymerase chain reaction (RT- PCR). Additionally or alternatively, expression of a gene may be inhibited by reducing or eliminating the translation of transcribed gene product(s) into a protein. A change in the level of translated gene products can be measured using any technique capable of detecting and/or quantifying proteins. Suitable methods are known in the art, and include, for example, immunohistochemistry, SDS-PAGE, immunoassays, proteomics and the like.

The expression of a gene may be inhibited by the introduction of one or more mutations into the nucleotide sequence of the gene. Suitable mutations include, but are not limited to, missense mutations, nonsense mutations, truncation mutations, insertion mutations, deletion mutations or any combination thereof.

By way of non-limiting example only, a target gene in a cell (i.e. a gene encoding a protein of a PEMT pathway) may be inactivated by inserting exogenous genetic material into the gene and/or substituting the gene (or a portion of the gene) with exogenous genetic material.

In certain embodiments, exogenous DNA is integrated into the chromosomal DNA (i.e. the target gene) of the cell by recombination (e.g. homologous recombination or heterologous recombination). For example, homologous recombination may be used to substitute a target gene/portion of a target gene in the genome of a cell with similar DNA derived from a vector construct. Accordingly, a vector construct comprising a target gene sequence (or a portion of a target gene sequence) having one or more mutations (e.g. those defined above) may be introduced into the cell. Homologous recombination may facilitate replacement of the target gene (or portion of the target gene) in the host cell genome with similar sequence (comprising mutation(s)) derived from the vector construct. Exogenous genetic material incorporated into the target cell genome by homologous recombination inhibits expression of the target gene in the cell. It will be understood that a "target gene" includes regulatory sequences of that gene. Hence, the exogenous genetic material may be inserted into exonic/intronic sequences and/or regulatory sequences of the gene (e.g. promoter sequences and the like).

Cells in which the target gene is inactivated may be selected and propagated using techniques known in the art. For example, the genetic material incorporated may comprise a selectable marker (e.g. an antibiotic resistance gene) that facilitates target gene inactivation (by disrupting the coding sequence of the gene) and also allows the selection of cells in which the gene has been inactivated in the presence of an antibiotic.

Methods for the production of vector constructs suitable for introducing exogenous genetic material into a cell are generally known in the art and are described, for example, in Ausubel.et al. (Eds), (2007), "Current Protocols in Molecular Biology", John Wiley & Sons; ,

24

Sambrook et al., (2001), "Molecular Cloning: A Laboratory ManuaF, 3rd Ed., Cold Spring Harbor Laboratory Press; and Coico et al. (Eds), (2007), "Current Protocols in Microbiology ", John Wiley and Sons, Inc. The vector may be a plasmid vector, a viral vector, a phosmid, a cosmid or any other suitable vector construct. The vector may comprise expression control and processing sequences such as promoters, enhancers, polyadenylation signals and/or transcription termination sequences. The vector construct may also include a selectable marker, for example, an antibiotic-resistance gene such as chloramphenicol or tetracycline.

Genetic material for insertion into the construct (e.g. DNA with homology to a target gene comprising mutation(s)) may be generated, for example, by chemical synthesis techniques such as the phosphodiester and phosphotriester methods (see for example Narang et al, (1979), "Improved phosphotriester method for the synthesis of gene fragments ", Meth. Enzymol. 68:90; Brown et al, (1979), "Chemical synthesis and cloning of a tyrosine tRNA gene", Meth. Enzymol. 68: 109; and United States Patent No. 4356270), or the diethylphosphoramidite method (see Beaucage et ah, (1981), "Deoxynucleoside Phosphoramidites - A New Class of Key intermediates for Deoxypolynucleotide Synthesis ", Tetrahedron Letters, 22:1859-1862). Genetic material for insertion into the construct may be amplified in number, for example, by performing polymerase chain reaction (PCR) assays on DNA or cDNA sequences, or by performing RT-PCR on RNA sequences. The resulting nucleic acids may then be inserted into the construct, for example, by restriction-ligation reactions or by the TA cloning method.

Suitable methods for the introduction of vector constructs and other foreign nucleic acid material into cells are generally known in the art, and are described, for example, in Ausubel et al. (Eds), (2007), "Current Protocols in Molecular Biology ", New York: John Wiley & Sons; and Sambrook et al., (2001), 3rd Ed., "Molecular Cloning: A Laboratory ManuaF, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.

By way of example only, target cells may be transfecteS with vector constructs using the "heat shock" method. Under this method the cells are chilled in the presence of divalent cations such as Ca ²⁺ , which causes cell wall/membrane permeability. Cells are mixed with the construct, incubated on ice, and then briefly heat shocked {e.g. at 42 °C for 0.5-2 minutes) allowing the vector construct to enter the cell.

Alternatively, target cells may be transfected with vector constructs by electroporation, a method that involves briefly shocking the cells with an electric field causing the cells to ,

25 briefly develop holes through which the construct may enter the cell. Natural membrane- repair mechanisms rapidly close these holes after the shock.

Vector constructs may be introduced into fungal cells (e.g. protoplast cells) using methods described in Pentilla et al., (1987), " versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesef Gene, 61 : 155-164.

Following entry of the vector construct into a target cell, the cell may be cultured under conditions suitable to facilitate reproduction. Reproduction of the cell facilitates replication of genetic material including that of the construct. Reproduction of the cell may also facilitate recombination between genetic material in the construct and genetic material of the host cell genome. Methods for the culture of cells are known in the art and described in, for example, Coico, et al. (Eds), (2007), "Current Protocols in Microbiology ", John Wiley & Sons, Inc.

The culture may be performed in medium containing a substrate that facilitates the identification of cells comprising vector constructs and/or cells in which a target gene has been inactivated. Accordingly, target cells containing vector constructs and or inactivated target gene(s) may be selected and propagated. For example, if the vector contains one or more selectable markers, cells containing vector constructs may be identified by expression of the marker or markers in the presence of the relevant substrate. Using the example of a drug resistance gene such as an antibiotic resistance gene, cells containing vector constructs can be identified by their ability to grow and propagate in selection medium containing the corresponding antibiotic.

Additional sequences capable of enhancing the frequency of homologous recombination events may be included in the vector constructs. For example, eukaryotic or prokaryotic recombination enzymes such as REC A, topoisomerase, REC 1 or other DNA sequences which enhance recombination (e.g. Chi sequence) may be included in the constructs. Furthermore, sequences that enhance transcription of chimeric genes produced by homologous recombination may also be included in the vector constructs, non-limiting examples of which include inducible elements such as the metallothionine promoter. The cell may also be administered various other proteins known in the art to be capable of increasing recombination frequencies.

In certain embodiments, the expression of a gene in a cell is inhibited using an anti- sense nucleic acid to block the translation of polypeptides from RNA transcripts.

Anti-sense nucleic acids of the invention may be of at least five nucleotides in length and are generally oligonucleotides which range in length from 5 to about 200 nucleotides. For example, an anti-sense oligonucleotide of the invention may be at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175 or 200 nucleotides. The oligonucleotides may be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof. The oligonucleotides may be single-stranded or double-stranded. In certain embodiments, the oligonucleotides are small interfering RNA (siRNA) molecules.

An anti-sense nucleic acid of the invention may be modified at any position on its structure using substituents generally known in the art. For example, the anti-sense nucleic acid may include at least one modified base moiety which is selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5- carboxymemylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, 2,2-dimethylguanine, 2-methyl- adenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7- methylguanine, 5- methylaminomethyluracil, 5-memoxyaminomethyl-2-thiouracil, beta- D-mannosylqueosine, 5'-methoxycarboxymethyluracil, pseudouracil, 2-thiocytosine, 5- methyl-2-thiouracil, 2- thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5- oxyacetic acid (v), queosine, wybutoxosine, 5-methyl-2-thiouracil, 3- (3-amino-3-N-2- carboxypropyl) uracil, and 2,6-diaminopurine.

Anti-sense oligonucleotides, typically of 18-30 nucleotides in length (although longer or shorter length oligonucleotides are also contemplated) may be generated which are at least substantially complementary across their length to a region of the nucleic acid sequence of interest. Binding of the anti-sense oligonucleotide to a cellular nucleic acid comprising a complementary sequence may interfere with transcription, RNA processing, transport, translation and/or mRNA stability. Suitable anti-sense oligonucleotides may be prepared by methods well known to those of skill in the art, and may be designed to target and bind to regulatory regions pf the target nucleotide sequence, or, to coding (gene) or non-coding (intergenic region) sequences. Suitable anti-sense oligonucleotides may include modifications designed to improve their delivery into cells, their stability once inside a cell, and/or their binding to the appropriate target. For example, the anti-sense oligonucleotide may be modified by the addition of one or more phosphorothioate linkages, or the inclusion of one or morpholine rings into the backbone (so-called 'morpholino' oligonucleotides).

Double-stranded RNA (dsRNA) molecules may be synthesised in vitro in which one strand is identical to a specific region of the gene transcript of interest and then introduced into the target cell. Additionally or alternatively, corresponding dsDNA can be employed which is converted into dsRNA once presented intracellularly.

Anti-sense nucleic acids capable of inhibiting the expression of a target gene may be introduced into a cell using a replicable vector. The vector may remain episomal or integrate into the genome of the cell. By way of example only, double-stranded RNA expressing constructs may be introduced into a cell to interfere with accumulation of endogenous mRNA encoding the target gene.

Methods of synthesizing RNA molecules are known in the art and are described, for example, in Verma and Eckstein, (1998), "Modified oligonucleotides: synthesis and strategyor users", Annu Rev Biochem., 67:99-134. Single-stranded RNAs for annealing into dsRNA molecules can also be prepared by enzymatic transcription from DNA plasmids isolated from recombinant bacteria or from synthetic DNA templates.

In certain embodiments, RNA interference (RNAi) may be used to inhibit the expression of a target gene in a cell. The RNAi may be a hairpin RNAi. RNAi refers to a means of selective post-transcriptional gene silencing by destruction of specific mRNA by small interfering RNA molecules (siRNA). The siRNA is generated by cleavage of double stranded RNA, where one strand is identical to the message to be inactivated.

The strand capable of hybridising to a portion of an RNA precursor encoding a polypeptide of the invention will, in general, have sufficient sequence complementarity to the RNA precursor to mediate target-specific RNA interference (RNAi). Accordingly, the second (non-hybridising) strand of the dsRNA molecule will have at least about 50%, 60%, 70%, 75%, 80%, 85%, or 95% sequence identity to an RNA precursor encoding a target protein. Preferably, the sequence identity is at least 85% and most preferably 100%.

In applications involving RNA interference, the length of a dsRNA molecule provided herein may be 19-25 nucleotides in length, and more preferably 20-22 nucleotides in length. In certain embodiments, at least one strand has a 3 '-overhang of 1-5 nucleotides, more preferably 1-3 nucleotides and most preferably 2 nucleotides. The second strand may be blunt-ended or have up to 6 nucleotides 3' overhang.

RNAi techniques and methods for the synthesis of suitable molecules for use in RNAi and for achieving post-transcriptional gene silencing are known in the art (see, for example, Chuang et al, (2000), Proc Natl Acad Sci USA 97: 4985-4990; Fire et al, (1998), Nature 391 : 806-811; Hammond et al, (2001), Nature Rev, Genet. 2: 1 10-11 19; Hammond et al,

(2000) , Nature, 404: 293-296; Bernstein et al, (2001), Nature, 409: 363-366; Elbashir et al,

(2001) , Nature, 411 : 494-498; PCT publication no. WO 1999/32619; PCT publication no. WO 1999/49029; PCT publication no. WO 2001/29058; and PCT publication no. WO 2001/70949).

In accordance with the methods of the invention, anti-sense nucleic acids capable of inhibiting the expression of a target gene may be stably introduced and expressed in a cell (e.g. a plant cell or an algal cell) using a vector construct.

The vector may be a plasmid vector, a viral vector, a phosmid, a cosmid or any other vector construct suitable for the insertion of foreign sequences, introduction into cells and subsequent expression of the introduced sequences. In a preferred embodiment, the vector is an expression vector comprising expression control and processing sequences such as a promoter, an enhancer, polyadenylation signals and/or transcription termination sequences.

Methods for the production of vector constructs, the production of genetic material to include in vector constructs and the introduction of vector constructs into cells are generally known in the art and are described in the preceding paragraphs of this section.

A further means of inhibiting the expression of a target gene in a cell includes introducing catalytic anti-sense nucleic acid constructs, such as ribozymes, which are capable of cleaving mRNA transcripts and thereby preventing the production of wild type protein. Ribozymes are targeted to and anneal with a particular sequence by virtue of two regions of sequence complementarity to the target flanking the ribozyme catalytic site. After binding the ribozyme cleaves the target in a site-specific manner. The design and testing of ribozymes which specifically recognise and cleave sequences of interest can be achieved using techniques well known to those in the art (see, for example, Lieber and Strauss, (1995), "Molecular and Cellular Biology", 15:540-551 ).

Reducing protein activity.

Reducing the activity of a protein in a cell in accordance with the methods of the invention can be performed using methods known in the art.

It will be understood that "reducing" protein activity as contemplated herein encompasses any reduction of the activity of the protein compared to its normal/standard activity at any given condition including, but not limited to, complete loss of activity.

By way of non-limiting example only, the activity of a target protein (e.g. a PEMT pathway protein) may be reduced by administering an antagonist of the protein to the cell. The antagonist may inhibit one or more of the biological activities of the protein, for example, by preventing interaction(s) with other protein(s) in the cell. ,

29

Antagonists of target proteins may be generated using a number of techniques known to those skilled in the art. For example, methods such as X-ray crystallography and nuclear magnetic resonance spectroscopy may be used to model the structure of a target protein, thus facilitating the design of potential modulating agents using computer-based modelling. Various forms of combinatorial chemistry may also be used to generate putative antagonists.

An antagonist of a target protein can potentially be any chemical compound, non- limiting examples of which include amino acids, nucleic acids, peptide nucleic acids, lipids, polypeptides, carbohydrates, and nucleosides. Other non-limiting examples include peptidomimetics (e.g. peptoids), amino acid analogues, polynucleotides, polynucleotide analogues, nucleotides, nucleotide analogues, metabolites, metabolic analogues, and organic or inorganic compounds (including heteroorganic and organometallic compounds).

Large libraries of chemicals (i.e. putative antagonists) may be screened for antagonistic activity against a target protein or target protein(s) using high-throughput methods. Such libraries of candidate compounds can be generated or purchased from commercial sources. For example, a library can include 10,000, 50,000, or 100,000 or more unique compounds. By way of example only, a library may be constructed from heterocycles including benzimidazoles, benzothiazoles, benzoxazoles, furans, imidazoles, indoles, morpholines, naphthalenes, piperidines, pyrazoles, pyridines, pyrimidines, pyrrolidines, pyrroles, quinolines, thiazoles, thiphenes, and triazines. A library may comprise one or more classes of chemicals, for example, those described in Carrell et al, (1994), Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al, (1994), Angew. Chem. Int. Ed. Engl. 33:2061; Cho et al, (1993), Science 261 :1303-1305; DeWitt et al, (1993), Proc. Natl. Acad. Sci. U.S.A. 90:6909-6913; Erb et al, (1994), Proc. Natl. Acad. Sci. USA 91 :11422-11426; Gallop et al, (1994), J. Med. Chem. 37:1233-1251; and Zuckermann et al, (1994), J. Med. Chem. 37:2678-2685.

In general, putative antagonists may be screened for antagonistic activity against target protein(s) by contacting the putative antagonist with the protein(s) under conditions suitable for an interaction to occur.

Putative antagonists which bind, or otherwise interact with target protein(s) (or nucleic acids encoding a target protein), and specifically agents which inhibit their activity, may be identified by a variety of suitable methods. Non-limiting methods include the two-hybrid method, co-immunoprecipitation, affinity purification, mass spectroscopy, tandem affinity purification, phage display, label transfer, DNA microarrays/gene coexpression and protein microarrays. .

30

In certain embodiments of the invention, the activity of a target protein in a cell may be inhibited by an antibody specific for the protein.

An antibody that "specific for" a target protein is one capable of binding to the target protein with a significantly higher affinity than it binds to an unrelated molecule (i.e. a non- target protein). Accordingly, an antibody specific for a target protein is an antibody with the capacity to discriminate between the target protein and any other number of potential alternative binding partners. Hence, when exposed to a plurality of different but equally accessible molecules as potential binding partners, an antibody specific for a target protein will selectively bind to the target protein and other alternative potential binding partners will remain substantially unbound by the antibody. In general, an antibody specific a target protein will preferentially bind to the target protein at least 10-fold, preferably 50-fold, more preferably 100- fold, and most preferably greater than 100-fold more frequently than other potential binding partners that are not the target protein. An antibody specific for a target protein may be capable of binding to other non-target molecules at a weak, yet detectable level. This is commonly known as background binding and is readily discernible from target protein-specific binding, for example, by use of an appropriate control.

Preferably suitable antibodies are prepared from discrete regions or fragments of the target protein. An antigenic portion of a target protein may be of any appropriate length, such as from about 5 to about 15 amino acids. Preferably, an antigenic portion contains at least about 5, 6, 7, 8, 9, 10, 1 1, 12, 13 or 14 amino acid residues.

An antibody that binds specifically to a target protein can be generated using methods known in the art. For example, a monoclonal antibody specific for target protein, typically containing Fab portions, may be prepared using the hybridoma technology described in Harlow and Lane (eds.), (1988), "Antibodies-A Laboratory Manual", Cold Spring Harbor Laboratory, N.Y. In essence, in the preparation of monoclonal antibodies directed toward a target protein, any technique that provides for the production of antibodies by continuous cell lines in culture may be used. These include the hybridoma technique originally developed by Kohler and colleagues (see Kohler et ai, (1975), "Continuous cultures of fused cells secreting antibody of predefined specificity ", Nature, 256:495-497) as well as the trioma technique.

Screening for the desired antibody can also be accomplished by a variety of techniques known in the art. Suitable assays for immunospecific binding of antibodies include, but are not limited to, radioimmunoassays, ELISAs (enzyme-linked immunosorbent assay), sandwich immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, .

31 immunodiffusion assays, in situ immunoassays, Western blots, precipitation reactions, agglutination assays, complement fixation assays, immunofluorescence assays, protein A assays, Immunoelectrophoresis assays, and the like (see, for example, Ausubel et al., (1994), "Current Protocols in Molecular Biology", Vol. 1 , John Wiley & Sons, Inc., New York).

In certain embodiments, a vector construct capable of expressing an antagonist of a target protein may be used to reduce the activity of the target protein in the cell. Methods for generating vector constructs and administering them to cells are known in the art described in the section above entitled "Inhibition of gene expression ".

For example, a vector construct capable of expressing an antibody or antibody fragment specific for a target protein may be administered to the cell. Methods for the generation of such constructs and their expression in target cells are known in the art and exemplary methods are provided, for example, in US Patent no. 7112439 (issued on 26 September, 2006 to Johnson et at); US patent publication no. 2009-0104660 (filed on 23 April, 2009, Yung et al); Cabilly et al., (1984), "Generation of antibody activity from immunoglobulin polypeptide chains produced in Escherichia coh ^" ", Proc. Natl. Acad. Sci. USA 81 : 3273-3277, and Boss et al., (1984), "Assembly of functional antibodies from immunoglobulin heavy and light chains synthesised in E. coli", Nucleic Acids Res. 12: 3791-3806.

Energy storage

In accordance with the methods of the invention, increasing levels of phosphatidic acid in a cell may provide a means of generating large/supersized lipid droplets within the cell. Without limitation to a particular mode of action, it is postulated that increasing cellular levels of phosphatidic acid serves to enhance the fusion properties of intracellular lipid droplets resulting in the formation of large/supersized lipid droplets within the cell.

Fusing multiple lipid droplets with a cell to form large lipid droplet(s) may provide a means of modifying lipid storage within the cell. In particular, fusing multiple lipid droplets with a cell to form large lipid droplet(s) may be utilised as a means to improve storage of energy within the cell.

In certain embodiments, the cell may be a microorganism or plant, non-limiting examples of which are provided in the section above entitled "Microorganisms and plants".

The levels of phosphatidic acid may be increased in the cell using any suitable method, including but not limited to those described in the section above entitled "Phosphatidic acid production and the PEMT pathway". ,

32

Genetically modified microorganisms and plants

Certain aspects the invention relate to genetically modified organisms (e.g. microorganisms and plants) in which the production of a lipid (e.g. a glycerolipid) is increased compared to a corresponding wild-type organism. In general, the genetically modified organisms comprise at least one genetic modification that increases phosphatidic acid production.

The "genetic modification" may be any change to the genetic make-up of the organism, non-limiting examples of which include the introduction of mutation(s) into endogenous genetic material, the deletion of endogenous genetic material, and the introduction of exogenous genetic material (e.g. exogenous genetic material integrated into the genome of the cell; exogenous genetic material introduced into the cell by way of a construct such as a plasmid or virus; exogenous nucleic acids such as oligonucleotides, RNA molecules (including RNAi) and the like).

The genetic modification may be a permanent genetic modification (i.e. one that is stably inherited by progeny) or a transient genetic modification (i.e. one that is lost after one or more generations of progeny).

It will be understood that the genetically modified organisms of the present invention are non-human organisms.

A genetically modified organism in accordance with the invention may be any organism capable of lipid production, in particular glycerolipid production, and preferably triacylglycerol and/or sterol ester production.

For example, the modified microorganism may be a bacterial, algal, or fungal species, non-limiting examples of which are provided above in the section entitled "Microorganisms and plants ".

A genetically modified plant in accordance with the invention may be any plant capable of lipid production and in particular glycerolipid production. Non-limiting examples of such plants are provided above in the section entitled "Microorganisms and plants ".

Genetically modified organisms of the invention are capable of increased lipid production. It will be understood that "increased lipid production" in a genetically modified organism of the invention refers to the increased production of at least one type of lipid in the organism compared to the production of that same lipid (under the same biological conditions) in a corresponding organism that has not been genetically modified. This does not necessarily exclude decreased production of particular type(s) of lipid(s) in the genetically modified organism provided that the production of at least one type of lipid is increased. Preferably, the production of triacylglycerols and/or sterol esters is increased in genetically modified microorganisms and plants of the invention compared to a corresponding wild-type microorganism or plant. Preferably, the triacylglycerols and/or sterol esters comprise short and/or saturated acyl chains. Non-limiting examples of preferred triacylglycerols and preferred fatty acid chain components of the triacylglycerols and sterol esters are provided in the section above entitled "Lipid production ".

Genetically modified organisms of the invention may comprise any genetic modification that increases cellular production of phosphatidic acid. For example, the modification may inhibit the phosphatidylethanolamine N-methyltransferase (PEMT) pathway. It will be understood that a "genetic modification" as contemplated herein includes, but is not limited to, any modification to genetic material of the organism (e.g. DNA, RNA, cDNA and the like) and the introduction/expression of foreign genetic material in cell(s) of the organism. The invention also contemplates genetically modified organisms generated by selective breeding. Preferably, the genetic modification is of a permanent or semi-permanent nature such that it is inherited by offspring of the organism. Preferably, the genetic modification is of a permanent nature such that it is inherited by offspring of the microorganism or plant. It will also be recognised that although the modification may be introduced by direct genetic manipulation of a target gene in cells of the organism, selection and classical genetics involving sexual crosses may also be utilised in appropriate cases.

The invention provides modified microorganisms and plants having increased levels of phosphatidic acid production in their cell(s). Phosphatidic acid production may be increased in cell(s) of the microorganisms and plants by any suitable genetic modification.

For example, phosphatidic acid production may be increased by introducing a genetic modification that increases the quantity and/or activity of one or more proteins or compounds in a lipid biosynthetic pathway. Alternatively, phosphatidic acid production may be increased by introducing a genetic modification that decreases the quantity or activity of one or more proteins or compounds in a lipid biosynthetic pathway. Non-limiting examples of genes and combinations of genes that may be subjected to modifications to increase phosphatidic acid production are provided in the section above entitled "Phosphatidic acid production and the PEMT pathway".

In certain embodiments, the genetic modification inhibits the phosphatidylethanolamine N-methyltransferase (PEMT) pathway. Any genetic modification that inhibits the PEMT pathway may be utilised. Non-limiting examples of genes and combinations of genes that may be subjected to modification so as to increase phosphatidic acid production and/or _t

34 inhibit the phosphatidylethanolamine N-methyltransferase (PEMT) pathway are provided in the section above entitled "Phosphatide acid production and the PEMT pathway" .

In some embodiments of the invention, the PEMT pathway is inhibited in the modified microorganism or plant by introducing one or more genetic modification that reduces the expression of a gene encoding:

(i) phospholipid methyltransferase (EC 2.1.1.16);

(ii) phosphatidylethanolamine methyltransferase (EC 2.1.1.17);

(Hi) inositol- 1 -phosphate synthase (EC 5.5.1.4);

(iv) CDP-diacylglycerol synthase (EC: 2.7.7.41);

(v) casein kinase II (EC 2.7.11.1);

(vi) a beta regulatory subunit of casein kinase II (EC 2.7.11.1);

(vii) a transcription factor that positively regulates one or more components of the PEMT pathway - for example, Saccharomyces cerevisiae IN02 YDR123C and/or INO4/YOL108C, or a protein homologous to Saccharomyces cerevisiae IN02 YDR123C or INO4 YOL108C;

(viii) Saccharomyces cerevisiae RTC2/YBR147W, or a protein homologous to Saccharomyces cerevisiae RTC2/YBR147W;

(ix) Saccharomyces cerevisiae MRPS35/YGR165W or a protein homologous to Saccharomyces cerevisiae MRPS35 YGR165W;

(x) Saccharomyces cerevisiae FLD1/YLR404W, or a protein homologous to Saccharomyces cerevisiae FLD1/YLR404W;

(xi) a Brassica sp. plant CDP-diacylglycerol synthase protein comprising the sequence defined by SEQ ID NO:l ; a Ricinus sp. plant CDP-diacylglycerol synthase protein comprising the sequence defined by SEQ ID NO:6; a Glycine sp. plant CDP-diacylglycerol synthase protein comprising the sequence defined by any one of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20 or SEQ ID NO:21; or a Chlamydomonas sp. protein encoded by the gene C 30067, the gene comprising the sequence set forth in SEQ ID NO: 41 ;

(xii) a Brassica sp. plant casein kinase II protein comprising the sequence defined by SEQ ID NO: 2 or SEQ ID NO: 3; a Ricinus sp. plant casein kinase II protein comprising the sequence defined by SEQ ID NO: 8 or SEQ ID NO: 9; or a Glycine sp. plant casein kinase II protein comprising the sequence defined by any one of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27;

(xiii) a Brassica sp. plant protein comprising the sequence defined by SEQ ID NO:4; a Ricinus sp. protein comprising the sequence defined by any one or more of SEQ ID ,

35

NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16; or a Glycine sp. protein comprising the sequence defined by any one or more of SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36;

(xiv) a Ricinus sp. phosphatidylethanolamine methyltransferase protein comprising the sequence defined by SEQ ID NO: 7;

(xv) a Ricinus sp. phospholipid methyltransferase protein comprising the sequence defined by SEQ ID NO: 12; or a Glycine sp. phospholipid methyltransferase protein comprising the sequence defined by SEQ ID NO: 28.

(xvi) a transcription factor that positively regulates one or more components of the PEMT pathway from Ricinus sp., comprising the sequence defined by SEQ ID NO: 10;

(xvii) a Ricinus sp. protein comprising the sequence defined by SEQ ID NO: 11 ;

(xviii) any combination of two or more of (i)-(xvii)

In certain embodiments, the genetic modification inhibits a vesicle-associated membrane protein-associated protein (VAMP-associated protein).

Non-limiting examples of VAMP-associated proteins that may be inhibited include Saccharomyces cerevisiae SCS2/YER120W or a homologous protein of another microorganism or plant; or a VAMP-associated protein comprising a sequence defined in SEQ ID NO: 5, SEQ ID NO: 17, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO: 40.

Genetically modified organisms of the invention may be generated using standard methods known in the art.

For example, vector construct(s) encoding anti-sense nucleic acid(s) capable of inhibiting expression of a target gene may be introduced and expressed in cell(s) of the organism. Suitable anti-sense nucleic acids, vector constructs, and methods for their administration to cells are described above in the section entitled "Inhibition of gene expression ". Alternatively, the vector construct may encode an antagonist of a target protein. Methods for the generation of suitable antagonists and their introduction into cells are described above in the section entitled "Reducing protein activity ".

In certain embodiments, genetically modified organisms of the invention comprise one or more mutations in a gene encoding a protein of a starch biosynthetic pathway or a polyhydroxyalkanoate biosynthetic pathway. Suitable mutations include, but are not limited to, missense mutations, nonsense mutations, truncation mutations, insertion mutations, deletion mutations or any combination thereof. Mutation(s) may be introduced into a gene of .

36

an organism using any method known in the art. For example, mutation(s) may be introduced by administering a vector construct to cell(s) of the organism facilitating the exchange of host genetic material with exogenous genetic material of the vector using methods described in the section above entitled "Inhibition of gene expression ".

In certain embodiments of the invention, genetically modified organisms are generated by introduction of a suicide vector into cell(s) of the organism. As contemplated herein, a suicide vector is a vector that is unable to replicate in a particular host and is maintained only if it recombines into the host genome. The suicide vector may comprise sequences that are homologous to DNA sequences flanking the target gene, homologous to DNA sequences within the target gene, or both. In general, the target gene encodes a protein (e.g. an enzyme or transcription factor) of a starch or polyhydroxyalkanoate biosynthetic pathway, suitable examples of which are provided in the section above entitled "Phosphatidic acid production and the PEMT pathway".

Methods for the production of suicide vectors and their introduction into cells are well known in the art and are described, for example, in Ausubel, et al. (Eds), (2007), "Current Protocols in Molecular Biology", New York: John Wiley & Sons; and, Sambrook et al., (2001), 3rd Ed., "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Genetic material for insertion into the suicide construct may be generated, for example, using the methods described in the section above entitled "Inhibition of gene expression".

Cell(s) in which exogenous genetic material from the vector has been incorporated into genomic DNA may be selected and propagated. In the case where the cell is derived from a multicellular organism (e.g. a plant) a genetically modified organism may be generated from the cell using techniques known in the art.

The skilled addressee will recognise that the methods described above for inhibiting gene expression or inhibiting protein function in genetically modified microorganisms and plants of the invention are non-limiting examples, and that other suitable methods known in the art may also be utilised.

Biofuel production

Additional aspects of the invention relate to the production of biofuels from lipids. In certain embodiments, the biofuel is biodiesel. The lipids may be produced by a cell in accordance with the methods described herein. Alternatively, the lipids may be produced by a genetically modified organism (e.g. microorganism or plant) of the invention. ,

37

In general, the production of lipids from a genetically modified organism of the invention may be facilitated by cultivating the organisms under conditions suitable for their growth and/or reproduction. It will be understood that the term "cultivating" as contemplated herein encompasses any method of promoting the growth and/or, reproduction of an organism (e.g. algae, plants, bacteria, fungi). Methods for cultivating organisms such as algae, plants, bacteria, fungi are well known in the art.

Algae such as microalgae may be cultivated, for example, in closed or open ponds or photobioreactors. The growth rate of algae during cultivation may be regulated by varying factors including, but not limited to, light, temperature, salinity/pH of liquid medium, aeration (e.g. nitrogen and C0 ₂ exposure), photoperiod (i.e. light and dark cycles), and availability of nutrients (e.g. Ca(N0 ₃ ) ₂ ; KH ₂ P0 ₄ ; MgS0 ₄ ; NaHC0 ₃ , NaN0 ₃ , MgS0 ₄ , NaCl; K ₂ HP0 ₄ ; KH ₂ P0 ₄ , CaCl _2> Na ₂ HP0 _4> trace elements/metals).

Preferably, the lipid is a glycerolipid. More preferably, the lipid is a triacylglycerol. Non-limiting examples of preferred triacylglycerols and preferred fatty acid chain components of the triacylglycerols are provided in the section above entitled "Lipid production". In addition to triacylglycerols, lipids produced by a cell in accordance with the methods described herein and/or a genetically modified microorganism or plant of the invention may include, for example, free oils, fats, greases, diacylglycerides, and/or phospholipids.

In general, biofuel production methods of the invention comprise the step of isolating lipids from cells in which they are produced. Techniques for the extraction of lipids from cells (e.g. plant, algal and bacterial cells) are well known in the art. For examples, mechanical crushing (e.g. using an expeller or press) followed by chemical extraction using solvents such as aliphatics, supercritical liquids and gases, hexane, acetone, and primary alcohols, may be used to extract lipids from cells.

The methods of lipid extraction may incorporate additional biological agents to enhance the separation of materials and/or improve reaction rates. For example, various agents may be used to depolymerise algal/plant cell walls to assist in releasing lipids. Additionally or alternatively, viruses specific to specific algae (e.g. Chlorella Virus) and/or cyanophages (e.g. SM-1, P-60, and AS-1) may be utilised to rupture algal cell walls.

Algal and/or plant matter remaining after lipid extraction may be used as a source of biomass for alternative biofuel production processes (e.g. generation of bioalcohols via the separation and hydrolysis of starch followed by fermentation of short-chain sugars). ,

38

Lipids extracted in accordance with the invention may be treated using oxidative processes known in the art (e.g. ozonation, peroxone oxidation) to alter the degree of saturation of the lipids. This may increase the market value of the extracted lipids for biofuel production.

Extracted lipids can be further processed to produce biofuels.

In preferred embodiments, biodiesel is generated from extracted lipids. For example, acid esterification, base transesterification, and combinations thereof may be used to generate biodiesels from triacylglycerols. Transesterification is a process which includes reacting the triacylglycerol with alcohol or another alternative acyl acceptor to produce free fatty acid esters and glycerol.

Biodiesel may be produced via homogeneous base, acid, and/or enzyme catalyzed transesterification, as well as heterogeneous catalysed processes.

Base-catalysed transesterification may be used to transform triacylglycerols into alkyl esters (i.e. biodiesel). For example, an alcohol (e.g. methanol, ethanol, iso-propanol) mixed with a base such as potassium hydroxide or sodium hydroxide may be used to produce biodiesel from triacylglycerols using base-catalysed transesterification.

Additionally or alternatively, acid-catalysed transesterification may be used to transform triacylglycerols into biodiesel. For example, an alcohol (e.g. methanol, ethanol, iso-propanol) mixed with an acid (e.g. sulfuric acid) may be used to produce biodiesel from triacylglycerols using acid-catalysed transesterification. The rate of acid-catalysed transesterification may be enhanced by catalysing the reaction at increased temperature and/or pressure (e.g. more than 60°C and or more than 3 atmospheres)

Additionally or alternatively, triacylglycerols may be converted to biodiesel by direct hydrogenation.

In addition to the use of extracted triacylglycerols for biofuel production, extracted lipids (e.g. free oils, fats, greases, diacylglycerides and/or phospholipids) may be added directly to petroleum diesel as a blending agent.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. .

39

EXAMPLES

The invention will now be described with reference to specific examples, which should not be construed as in any way limiting.

Example 1: role for phosphatide acid in the formation of "supersized" lipid droplets

Materials and Methods (i) Yeast strains

S. cerevisiae wild type strain BY4741 (MATa; his3Al; leu2A0; metlSAO; ura3A0) and its derived non-essential gene-deletion strains were either obtained from EUROSCARP or generated in this study. The latter included pah J A (PAH1 HIS3MX6), and dgaJAlrolA (DGAl ::kanMX4, The Tet-promoter strains used for expression of essential genes under the regulatable Tet07 promoter were obtained from Open biosystems.

(it) Reagents

Yeast extract, peptone, dextrose, and yeast nitrogen base were purchased from BD. Nile red, choline, ethanolamine, inositol, doxycycline, cerulenin, and Ficoll 400 were from Sigma. 1, 2-dioctanoyl-sn-glycerol 3-phosphate (PA 8:0/8:0), 1, 2-dioleoyl-sn-glycero 3- phosphate (PA 18:1/18:1), oleoyl-L-a-lysophosphatidic acid, 1, 2-dioctanoyl-sn-glycerol (DAG 8:0/8:0), phosphatidylethanolamine, phosphatidylcholine, and triolein were purchased from Avanti Polar Lipids.

To screen for yeast mutants that generate supersized LDs, cells were cultured in synthetic complete media (0.67% yeast nitrogen base, 2% dextrose, and amino acids) in 96- well plates at 30°C till stationary phase. For Tet07-regulated strains, 15μg/ml doxycycline was added to repress specific genes. Cells were stained with 20 μ¾Ίη1 Nile red for LDs and observed by fluorescence microscopy. Yeast strains found to contain SLDs were recultured in synthetic complete media with aeration in 10 ml culture tubes to confirm the phenotype. These strains were also grown in YPD media (1% yeast extract, 2% peptone, and 2% dextrose) to examine the morphology of LDs.

For phospholipid precursor treatment, synthetic complete medium was supplemented with 1 mM choline, 1 mM ethanolamine, or 75 μΜ inositol. (Hi) Fluorescence microscopy

Fluorescent imaging was performed under a Leica CTR5500 microscope (Wetzlar, Germany) with an EL6000 fluorescent lamp. Images were taken with a DFC300 FX digital camera and a Leica LAS AF software. Yeast cells were viewed under a x 100/1.30 oil immersion objective lens. A 450-490-nm bandpass excitation filter, a 510 dichromatic mirror, and a 515-nm longpass emission filter (Leica filter cube 13) were chosen to observe Nile red- stained LDs. For statistical presentation of the percentage of cells containing supersized LDs, 200 cells were observed and percentage was calculated. The experiments were done in triplicates and the result was shown as mean ± SD. Mammalian LDs were stained with Bodipy 493/503 (Invitrogen) and observed with a 470/40-nm bandpass excitation filter, a 500-nm dichromatic mirror, and a 525/50-nm bandpass emission filter (Leica filter cube GFP).

To observe and record LD fusion, 3μ1 of mid-log phase cells (OD6oo~ 5) or purified LDs were stained with Nile red, spotted on a slide and covered with a coverslip. Under the microscope, cells in which two or several LDs lay close together were targeted. Images were collected at 0.5 second intervals.

(iv) Transmission electron microscopy

Cells were grown in rich medium until stationary phase, harvested, fixed with 2.5% glutaraldehyde and postfixed with 2% (w/v) osmium tetroxide. The samples were subsequently dehydrated in a series of graded ethanol and embedded in Spurr's Resin. 80nm ultrathin sections were stained with uranyl acetate and lead citrate and examined under a JEM- 1230 Joel electron microscope.

Total RNA was extracted using the RNeazy Plus kit (QIAGEN). cDNA was generated from total RNA using a Superscript VILO cDNA Synthesis Kit (Invitrogen). PCR reaction was performed using Rotor-Gene RG-3000A (Qiagen). Threshold cycle value for each gene was acquired at the log phase and gene expression was normalized to reference genes as indicated. For Affymetrix Array processing and analysis, samples were prepared according to the Affymetrix GeneChip® Yeast Genome 2.0 Array protocol. Differences between WT and fldlA strains were compared using one-way ANOVA and adjusted for false discovery rate at 0.05 level. Array data is deposited on Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/).

(v) Isolation of microsomes ,

41

Microsomes were isolated as described in (see Rieder and Emr SD, (2001) "Isolation of subcellular fractions from the yeast Saccharomyces cerevisiae", Curr Protoc Cell Biol Chapter 3: Unit 3 8). Briefly, WT and fldl cells were cultured in SC media till log phase (OD600-1.0) and harvested. Cells as 0.1 g (wet weight) /ml in 0.1 M Tris S04 were sequentially incubated in pH9.4/10 mM DTT for 10 min, and in 1.2 M sorbitol/20 mM Tris Cl, pH7.5/lx SC medium/zymolase 100 T (15 mg ml) for 30 min. Spheroplasts were lysed in HEPES lysis buffer (10 mM HEPES/KOH, pH6.8/50 mM potassium acetate/100 mM sorbitol/2 mM EDTA) with the aid of a Dounce homogenizer. After removal of cell dedris, lysates were centrifuged at 30 000 g for 10 min at 4°C. P30 000 g membrane pellets were resuspended in HEPES lysis buffer, loaded onto 1.2 M/1.5 M sucrose (prepared in HEPES lysis buffer) gradients, and centrifuged at 100 000 g for 1 h at 4°C. ER membranes were collected at the 1.2 M/1.5 M sucrose interface.

(vi) Isolation of lipid droplets

Lipid droplets were isolated ¾s described in Fei et al. (Fei et al., (2008), "Fldlp, a functional homologue of human seipin, regulates the size of lipid droplets in yeast", J Cell Biol 180: 473-482).

(vii) Isolation and analyses of lipids.

Lipids were extracted from zymolase-digested lyophilized yeast cells, isolated ER microsomes or lipid droplets. Briefly 900 μΐ ice-clod chlorofom methanol (1 :2) was added to samples. Mixtures were vigorously vortexed for 1 min, and incubated for 2 h in vacuum container with rotary shaking at 4°C. Then 400 μΐ ice-cold water and 300 μΐ chloroform were added, vortexed and incubated on ice for 1 min. After centrifugation at 12000 rpm for 3 min at 4°C, the lower organic phase was collected. Subsequently, 50 μΐ 1 M HC1 and 500 μΐ chloroform were added to the remainder, vortexed, and incubated on ice for 3 min. The lower organic phase was also collected after centrifugation at 12000 rpm for 3 min at 4°C, and combined with the first extract. The extracted lipids were blown dry with nitrogen gas, and resuspended in solvent for mass spectrometry analysis.

Lipidomic analysis, and quantitative measurement of neutral lipids via thin layer chromatography (TLC) were performed as described in Fei et al. (Fei et al., (2008), "Fldlp, a functional homologue of human seipin, regulates the size of lipid droplets in yeast", J Cell Biol 180: 473-482) and Low et al. (Low et al., (2008), "Caspase-dependent and -independent .

42 lipotoxic cell-death pathways in fission yeas t J Cell Sci 121 : 2671-2684). TLC plates were developed in chloroform/methanol/water (65:25:4) to separate phospholipid species.

(viii) In vitro fusion assay of artificial lipid droplets.

To prepare lipid emulsions, lipids were mixed in chloroform/methanol (2:1), dried under a stream of N2, resuspended in buffer (150 mM NaCl, 50 mM Tris/HCl, pH 7.5, 1 mM EDTA,), and sonicated. For emulsions, the molar ratio of TAG to total phospholipids was 2:2.5. Contaminating vesicles were removed, and LDs were concentrated by ultracentrifugation at 100,000 g .for 15 min. For light scattering, lipid concentration was 25 mM phospholipids and 20 mM TAG before centrifugation.

(ix) Statistical analysis

All data are presented as mean ± SD. Statistical comparison between the two groups was performed using Student's t-test. Microarray data were analyzed using one-way ANOVA and adjusted for false discovery rate at 0.05 level.

Results and Discussion

(i) Identification of additional yeast mutants with "supersized " LDs

A yeast mutant gene was identified fldlA that was observed to developed very large LDs (Figure 1A). Whereas the diameter of LDs in wild type (WT) cells typically ranges between 0.3 to 0.4 μπι, and rarely exceeds 0.5 μπι (see Czabany et al. (2008), "Structural and biochemical properties of lipid particles from the yeast Saccharomyces cerevisiae", J Biol Chem 283: 17065-17074), fldlA cells often synthesize LDs with a diameter larger than 1.0 um. LDs with a diameter greater than l.O _. um are arbitrarily described, as "supersized" LDs (SLDs), whose volume is over 30 times the average of wild type LDs. About 20% of fidl cells cultured in rich (YPD) medium contained SLDs, and the percentage increased to ~70% when cells were grown in minimal (synthetic complete/SC) medium (Figure 1A and Table

1).

Table 1. Yeast gene deletions that lead to the formation of supersized LDs (SLDs).

% of cells with

SLD ORF Gene Function SLDs*

SC YPD ,

43

media media

1 YLR404W FLD1, Unknown 66.1±1.0 22.3±3.7

SEI1

2 YGR157W CH02 PE methyltransferase 60.9±5.8 0.5±0,5

3 YJR073C OPI3 Phospholipid methyltransferase 89.3±3.2 0.7±0.3

4 YDR123C IN02 Transcription factor 97.0±1.0 0.3±0.3

5 YOL108C IN04 Transcription factor 96.3±1.6 0.5±0.0

6 YBR147W RTC2 Unknown 71.0±2.7 0.7±0.3

7 YGR165W MRPS35 Unknown 69.3±5.1 0.3±0.3

8 YGL019W CKB1 Beta regulatory subunit of casein 32.8±3.4 0.5±0.5 kinase 2

9 YOR039W CKB2 Beta' regulatory subunit of casein 20.0±3.2 0.7±0.3 kinase 2

10 YBR029C CDS1 CDP-DAG synthase 46.6±3.7 29.1±5.6

WT 0.7±0.3 0.3±0.3

Cells were grown in synthetic complete (SC) media or rich YPD media to stationary phase, stained with Nile red, and observed under a fluorescence microscope for LDs. Experiments were done in triplicates and ~200 cells were counted for all strains each time. The percentages of cells that displayed SLDs were represented as mean±SD. SC has 11 μΜ inositol because it contains yeast nitrogen base.

Given the effects various nutrients may have on the dynamics of LDs, it was hypothesised that growing cells on defined, minimal media (SC) would reduce the impact of nutrients and uncover additional genes. The entire collection of viable yeast deletion mutants (-4800) grown on minimal (SC) media for SLDs was screened. In order to identify essential genes impacting LD size, the collection of mutants where all essential genes are controlled by the TetOrpromoter, which can be switched off efficiently, were included (see Mnaimneh et al. (2004) "Exploration of essential gene functions via titratable promoter alleles", Cell 118: 31-44). Besides fldlA, nine additional mutants (sld2-10) were identified that produced supersized LDs (SLDs) (Figure 1 and Table 1). Except for two previously uncharacterized genes (R TC2ISLD6&MRPS35/SLD 7), the majority of the SLD genes appear to function directly or indirectly in the metabolism of phospholipids, especially phosphatidylcholine (PC). PC is synthesized in yeast via two pathways: the Kennedy pathway and the phosphatidylethanolamine N-methyltransferase (PEMT) pathway (see Figure 10). In the .

44

PEMT pathway, phosphatidylethanolamine (PE) is methylated to PC in three steps by two methyltransferases, Cho2p and Opi3p. Besides cho2A and opi3A mutants, ino2A and ino4A mutants are also defective in PC synthesis via PE methylation since Ino2p and Ino4p are transcription factors that positively regulate the PEMT pathway. When ckilA, pctlA, cptlA, cho2A, opi3A, ino2A, and ino4A cells were cultured in rich (YPD) medium, none of these mutants accumulated SLDs. In contrast, when grown in SC medium (no choline and hence little Kennedy pathway activity), approximately 60% of cho2A cells, 90% of opi3A cells, 97% of ino2A and ino4A cells produced SLDs, whereas ckilA, pctlA, and cptlA cells did not (Figure 1B&E). From these results, PC synthesis does appear to be critical in regulating the size of LDs. Interestingly, of the 825 essential genes examined, SLDs were observed only upon knocking-down CDS1 (encoding CDP-diacylglycerol synthase) (Figure 1D&E). Therefore, the synthesis of not only PC, but also other phospholipids could be important for LD growth. The SLDs observed in fldlA, cho2A, opi3A, ino2A, ino4A, and TetOrCDSl (thereafter referred to as cdsl when repressed by doxycycline) strains were further confirmed by electron microscopy (Figure IE). The levels of TAG and SE of all mutants were also examined, and the level of TAG was significantly increased in all mutants (Figure IF).

(ii) Defective TAG mobilization of "supersized" LDs

As compared to many small LDs in WT cells, the formation of SLDs limits the surface area that is accessible to lipases. Therefore, the mobilization of TAG may be impaired in sld mutants. TAG breakdown in fldlA, ino4A, and cdsl strains was monitored in the presence of 10 mg/L cerulenin that prevents their de novo synthesis (see Kurat et al., (2009), "Cdkl/Cdc28-dependent activation of the major triacylglycerol lipase Tgl4 in yeast links lipolysis to cell-cycle progression", Mol Cell 33: 53-63. These results show that TAG mobilization in the mutants is significantly slower than that of WT (Figure 11).

(Hi) Treatment of different phospholipid precursors exerted distinct effects on the formation ofSLDs

The finding that YPD media invariably decreased the percentage of cells displaying SLDs in all sld mutants suggested that certain components present in rich YPD media, but absent or low in SC media, suppressed SLD formation. Considering that Cdslp, Cho2p, Opi3p, Ino2p, and Ino4p are either enzymes or transcription factors involved in phospholipid biosynthesis, it was speculated that these components might be precursors of phospholipids. To examine this possibility, WT and mutant strains were cultured in SC media supplemented with 1 raM choline, 1 mM ethanolamine, or 75 μΜ inositol. Interestingly, inositol treatment reduced the SLD formation in all mutants (Figure 2). In contrast, ethanolamine addition had an opposite effect; it enhanced SLD formation in most of the mutants. As expected, choline addition completely blocked the formation of supersized LDs in cho2A, opi3A, ino2A and ino4A strains (Figure 2A&B), since exogenously added choline restored PC synthesis in these mutants through the Kennedy pathway. Surprisingly it also had similar effect in rtc2A and mrps35A strains, suggesting that these two genes may also function in PC metabolism. Choline addition also partially inhibited SLD formation in cdsl, ckbJA, and ckb2A cells, but had little effect in fldlA cells.

(iv) A link between the generation of "supersized" LDs and an elevated level of intracellular phosphatidic acid (PA)

One notable common feature among cho2A, opi3A, ino2A, ino4A and cdsl mutants is the accumulation of PA (Figure 3A). PA is a cone-shaped lipid that alters the curvature of the membranes, promotes both SNARE-dependent and -independent membrane fusion events, and is implicated in the assembly of lipid droplets from newly synthesized TAGs. To examine whether PA is a key player in the formation of SLDs, we first analyzed the cellular level of PA in the SLD mutants by LC-MS. Indeed a significant elevation of PA was seen in all mutants except fldlA cells, where the level of PA is only moderately elevated (Figure 3A&B). Inositol treatment reduces the cellular PA pool through increased synthesis of phosphatidylinositol (PI) and also through the activation of a Mg^-dependent PA phosphatase. Consistent with the implication of PA in SLD formation, inositol treatment resulted in a significant reduction of SLD formation in all mutants including fldlA (Figure 2). In addition, when two PA phosphatases PAH1 and DPPl) were overexpressed under a GAL1 promoter, both greatly reduced SLD formation in ino2A and ino4A cells, and also in fldlA cells (Figure 3C). Overexpression oiPAHl and DPPl did not change the level of PC, PE, PS and PI, but significantly reduced the cellular level of PA in fldlA cells (Figure 12). These results imply that the increased amount of PA may account for the formation of SLDs, and that the level of PA in subcellular organelles such as the endoplasmic reticulum where LDs originate may have changed in fldlA cells, despite an insignificant increase in overall PA in this mutant (see below).

(v) Fldlp could regulate the metabolism of phospholipids SLDs in yeast were originally identified in fldlA cells; but the molecular function of Fldlp/seipin remains elusive (Fei et al., (2008), "Fldlp, a functional homologue of human seipin, regulates the size of lipid droplets in yeast", J Cell Biol 180: 473-482). To gain more insights into the function of Fldlp, mRNA microarray analysis was performed in WT and fldlA cells. Of ~5800 transcripts examined, INOl and OPI3 were the only transcripts whose levels were significantly upregulated in fldlA cells (Figure 4A). Quantitative real-time PCR confirmed a ~5-fold increase in the INOI mRNA level in fldlA cells (Figure 4B). INOl gene expression is derepressed when intracellular PA concentration rises. It was therefore examined the level of PA on the ER where the Opilp-Scs2p regulatory complex of INOl expression exists. Indeed, a significant increase of PA was observed in microsomes isolated from fldlA cells (Figure 4C). These results show that Fldlp, as other SLD mutants, can regulate PA metabolism, and also suggest that both the level and location of PA are relevant to droplet formation.

(vi) Elevated PA enhances SLD formation in yeast

Another strategy to increase PA is through inactivation of Pahlp, the PA phosphatase and ortholog of mammalian lipin proteins. Deletion of PAH1 leads to a dramatic increase in the level of its substrate, PA, but causes a dramatic reduction in the amount of TAG. Although the number of LDs was significantly reduced in pahlA cells, LD size was comparable to that in WT cells. Remarkably, SLDs were detected consistently in -3% of pahlA cells, though its TAG synthesis was decreased by over 50% (Figure 5A-C). In contrast, no SLDs were ever observed in dgalA IrolA cells, which have little diacylglycerol (DAG) acyltransferase activity. Interestingly, when pahlA cells were supplemented with oleate and DAG which bypasses the lack of PA phosphatase activity, the number of cells producing SLD increased to -30%, whereas oleate alone had no effect (Figure 5D). These results further indicate that PA plays an important role in SLD formation, and this role is more pronounced when the biosynthesis of TAG is not severely compromised.

(vii) Elevated phosphatidylethanolamine concentration is a factor in the biogenesis of SLDs The result that ethanolamine addition enhanced SLD formation in nearly all mutants

(Figure 2) suggested that an elevated phosphatidylethanolamine (PE) concentration could have a role in SLD formation, given that PE is also a cone-shaped phospholipid that can increase membrane curvature, thereby promoting LD monolayer coalescence. Consistent with this notion, mutants that accumulate PE also displayed a higher percentage of cells forming SLDs, particularly opi3A, ino2A, and ino4A (Table 1). As shown in Figure 6A, the lipidomic analysis further revealed that lipid droplets isolated from cho2A, ino2A, and ino4A also had a higher PE to PL (total membrane phospholipids) ratio than those of WT cells. In addition, ethanolamine treatment significantly increased the proportion of PE on lipid droplets of fldlA and cdsl cells. Even in LDs of ino4A cells, ethanolamine addition still moderately increased the PE to PL ratio, though its PE level was already much higher than WT. However, elevated PE alone was not able to induce SLD formation since inositol addition completely abolished the biogenesis of supersized LDs despite that a higher PE to PL ratio persisted in ino4A cells (Figure 6A). Moreover, SLDs were abundant in cdsl where the PE/PL ratio was lower than that of the WT (Figure 6A &13).

(viii) Reduced PL to TAG ratio does not completely correlate with the formation of SLDs

A decreased phospholipid (PL) to TAG ratio could also induce SLD formation, since coalescence may be induced to decrease the surface-to-volume ratio of droplets when phospholipids are limiting. This model appears to be true for the mutants grown in SC media. However, when grown in YPD media, SLDs disappeared in cho2A, ino2A and ino4A strains but the decreased PL to TAG ratio persisted (Figure 6B). In addition, inositol supplementation did not increase the phospholipid to TAG ratio in cdsl or ino4A mutant (Figure 6B).

(ix) Rtc2p and Mrps35p, new players in phospholipid metabolism

Choline addition completely inhibited SLD formation in rtc2A and mrps35A, in a manner similar to cho2A, opi3A, ino2A, and ino4A, strains known to be defective in the methylation of PE into PC (Figure 2). This phenotype suggests that deletion of RTC2 or MRPS35 might affect PC synthesis through the PEMT pathway. As expected, lipidomic analysis revealed that rtc2A and mrps35A strains displayed a 2.5-fold increase of PE to PC ratio, indicating these two gene products are involved in PC synthesis through the PEMT pathway (Figure 7). rtc2A and mrps35A cells . also synthesized ~60% more phosphatidylinositol (PI) than WT, possibly resulting from the accumulation of CDP-DAG due to a blocked PEMT pathway (Figure 7&13A). The phospholipid profiles of ckblA and ckb2A strains were also examined and found that both synthesized less PC and PE than WT without causing significant changes in the PE to PC ratio (Figure 7&13A).

(x) LDs ofsld mutants demonstrated enhanced fusion activities both in vivo and in vitro .

48

It was next investigated how changes in phospholipids in the sld mutants may lead to the formation of SLDs. One possibility could be enhanced fusion activities. Indeed, fusion of Nile red-stained LDs could be observed in cho2A, opi3A, ino2A, ino4A, and cdsl strains (Figure 8), and also in rtc2A, mrps35A, ckblA and ckb2A strains (data not shown). The fusion frequency of LDs in each mutant was similar (-10 out of 200 adjacent pairs of LDs) to that of fldlA . Furthermore, LDs isolated from representative strains demonstrated fusion activities in vitro (videos S3 and S4). It should be noted that no fusion events in wild type yeast cells were ever observed with the methods employed.

(xi) Phosphatidic acid induces fusion of artificial LDs in vitro

To obtain direct evidence that PA induces coalescence of small LDs to form supersized droplets, artificial LDs were made and their stability tested. After generating the artificial droplets by sonication, we removed liposomes formed at the same time by density gradient centrifugation. This fractionation also concentrated the artificial droplets. From this starting point, we followed the stability of LDs by light scattering, which directly measures the number of LDs. When the concentration of PA in artificial LDs was increased, their number decreased significantly during incubation (Figure 9). In the presence of PE, a smaller fraction of PA (-3% molar ratio, PC:PE:PA 3:1 :0.13) achieved a similar effect compared to the coalescence observed in the presence of PC covered LDs (~5% PA (PC:PA 20:1). These results show that PA reduces the stability of LDs and mediates coalescence of LDs, and that this property of PA may be modulated by the phospholipid composition of LDs.

(xii) Discussion

Lipid droplets are dynamic organelles whose number and size undergo constant changes in response to internal and external cues. The physiological relevance of the size of the LDs is not well understood, and far less is known about how the size of LDs is determined at the molecular level. In this study, key proteins were identified that govern the size of LDs in yeast by modulating phospholipid metabolism. Also identified were proteins previously unknown to regulate phospholipid metabolism. Most importantly, in vivo and in vitro evidence is provided that phosphatidic acid can influence the size of the LDs.

SLDs provide an efficient form of fat storage in terms of surface to volume ratio. An interesting question is whether cells automatically generate SLDs upon lipid loading to economize on the synthesis of phospholipids that form the surface of lipid droplets. While large lipid droplets are typical in white adipocytes, most other cell types (brown adipocytes, hepatocytes, myocytes) store lipids in numerous small LDs. In WT yeast cells, a dramatic .

49 increase in the number but not size of LDs is often observed in growth conditions favoring neutral lipid synthesis and storage, such as starvation. Maintaining small LDs may be physiologically important: upon starvation, yeast cells convert phospholipid intermediates and sterols to neutral lipids which in turn can be hydrolyzed to release fatty acids and sterols for immediate membrane synthesis and cell growth when glucose becomes available. Lipolysis may be important for efficient cell-cycle progression in yeast and lipolysis occurs more efficiently for small LDs (Figure 11). Therefore, it appears that SLDs are only formed in highly specialized, non-dividing cells (e.g. fully differentiated white adipocytes) or under pathological conditions such as severe hepatic steatosis.

Genetic factors that regulate the size of LDs were identified in a screen of yeast. Decreased PC synthesis, and consequently an increased PE to PL ratio (or a decrease in PC/TAG), has been associated with SLD formation (Figure 1). LDs are phase-separated organelles in the cytoplasm. Thus, unlike for other organelles, the steady state and lowest energy state is probably to have only one droplet (this would minimize interfacial surface energy). Some phospholipids (specifically PC) shield droplets from coalescence. Fusogenic lipids such as PA and PE could overcome this effect. In agreement with this notion, it was identified that PE and PA both have an effect on SLD formation. First, treatment of ethanolamine further increased PE to PL ratio and enhanced SLD formation in most mutants (Figures 2&6); in addition, strains with increased levels of both PE and PA also had a higher percentage of cells producing supersized LDs (Table 1). Decreased levels of PLs also could lead to SLD formation, since phospholipids levels may not be sufficient under these conditions to prevent the hydrophobic TAG phases to fuse. If the amount of PLs on LDs is not sufficient, fusion would occur until the surface-to-volume ratio of LDs is reflecting the ratio of phospholipids to TAG. At this point the monolayer would shield the LD from any further fusion.

Identification of the cdsl mutant through the screening of the knock-down collection of essential yeast genes brought attention to PA, whose critical role in SLD formation was confirmed by the strong "size-reduction" effect of inositol supplementation, an efficient and reliable way to reduce PA in yeast. The essential role of PA in SLD formation was further confirmed when all mutants that develop SLDs were found to accumulate PA (Figure 3), including the seipin-deficient (fldlA) (Figure 4) and lipin-deficient (pafilA) (Figure 5A&B) cells. PA is a central intermediate in the synthesis of major glycerolphospholipids and TAG, as well as an important signaling lipid. Different pools of PA and distinct PA subclasses may account for the diversity of PA function. For instance, the yeast Opilp (ER localized transcription repressor) senses only a PA pool on the ER but not the plasma membrane PA pool regulated by the yeast phospholipase D Spol4p. Deleting or overexpressing SP014 did not have any impact on the formation of SLDs (data not shown), suggesting that an intracellular (ER/LD) PA pool is responsible for SLD formation. This appears to be also the case for the fldlA mutant, in which the level of microsomal PA, but not overall PA, is significantly increased. The level of PA on the ER could very well reflect the amount of PA on the LDs, given that LDs are believed to originate from the ER. In summary, it was found that increased PA levels may overcome the effect of phospholipid shielding, and that the location of PA also matters.

Besides establishing a strong link between PA and the size of LDs, these results provided herin also reveal that Rtc2p and Mrps35P can regulate the PEMT pathway of PC synthesis. Both Rtc2p and Mrps35p associate with mitochondria. Exactly how ckblA and ckb2A mutants cause a significant increase in cellular PA and thereby the formation of SLDs is not clear, although the key transcription factor that regulates phospholipid synthesis in the yeast, Opilp, can be phosphorylated and regulated by casein kinase 2.

Finally, the mechanistic link between changes in the level of PA/PE and the formation of SLDs was investigated. LDs are covered by a monolayer of phospholipids, whose composition may have a profound effect on the dynamics of the LDs. Both PA and PE are cone-shaped, fusogenic lipids that can alter the curvature of the membranes. PA, in particular, promotes both SNARE dependent and independent membrane fusion events. It is possible that a higher level of PA on the monolayer of the LDs would promote spontaneous fusion of contacting LDs. Indeed, in vivo microscopic observation found increased incidents of LD fusion in mutants with increased level of PA (Figure 8). Remarkably, when artificial LDs are made with different ratios of PA, PE and PC, it is clear that even a small amount of PA could significantly increase the size of the LDs (Figure 9).

In summary, studies described herein identify novel protein and lipid regulators of the size of the LDs, an important lipid-storage organelle. Knowing how LD size is determined may provide invaluable insights into how human cells/tissues handle abnormal influx of lipids in today's obesogenic environment. Example 2: Knockdown of gene expression of CDSlp homologue in the green alga Chlamydomonas increases lipid droplet accumulation

Materials and Methods

(i) Algal strains

Chlamydomonas reinhardtii wild type strain 21gr.

(ii) RNAi Construct and transformation

A hairpin RNAi construct was made by using 1 ^st -3 ^rd exon of the Chlamydomonas homologue of CDSlp driven by LC8 promoter. The gene name of the homologue is C 30067. The LC8 promoter is from Chlamydomonas.

The sequence of the first three exons of the gene is the following:

ATGCGACCAAAACAACAAGCTCAGGTCACCGCCGCGGAGGACACCGCGTCGGAGCCG GAGCCGGA GCCCAAGCCCGTGGATCGAAAGTCGAGGCTCAGGTCCTTCCGCGTACGCACCATCTCCTC GGTCT TGCTCATCGGCGGGTTCATCGGCATCATTTGGGCGGGTCATGTGCCGCTTATGTTCTTCA TCCTG CTACTCCAGTTCCTGGTGGCCCGGGAGCTCTTCCGCATCGCCTACATAGCGGAGAAGTCC AAACG CAGCC ( SEQ ID NO : 41 )

This construct was transformed into Chlamydomonas using a glass-bead method. The selection marker for transformation was APHVIII, whose expression conferred resistant to paromomysin.

(Hi) RNAi strain selection

The survival strains on agar plates were screened by using Nile Red staining, lug ml final concentration of Nile red was used.

(iv) Microscopic observation

A Zeiss fluorescence microscope (Observer.Zl) with GFP filter was used for screening putative RNAi strains. ,

52

Results

It was observed that Chlamydomonas reinhardtii transformed with the CDS1 gene RNAi accumulated lipid droplet drastically. Figure 14 shows typical fluorescent images of wild type (A - left image) and the RNAi strain (B - right image).

Summary

CDSlp is a key enzyme in lipid metabolism. To understand whether it plays a role in lipid droplet accumulation in algae, expression of the CDSlp homologue in Chlamydomonas was knocked down and droplet accumulation analyzed. It was observed that lipid droplet increases drastically in the RNAi strains.

Example 3: identification of genes in Brassica sp. (mustard), Ricinus sp. (Castor Bean), and Glycine sp. (soybean) homologous to S. cerevisiae genes

L Brassica sp. (mustard) vs S. cerevisiae alienments:

* Cdsl

SEQ ID NO: 1 Brassica sp. XA_0051 (Cdsl homologue)

DKNKYRSl^IRTCSSLWMLGGWFIIYMGHLYI AMWGIQIF AKELFFLLRRAHEERRLPGFRLLNWHFFFTA MLFWGRILQQQLWTVSSDRFIYKLVSGLIKYQMVICYFLYIAGFMWFILTLKKK YKYQFGQYAW HMILIW FTQSSFTVANIFEGIFWFLLPAALIAMNDVAAYFFGFYFGKTPLIKLSPKKTWEGFIGA

>XA_ )051 Length - 1633677

E-value = 1.37e-34 ₍ Score = 367, Bitscore = 145.976,

Identities = 77/209 (36%), Positives = 122/209 (58%), Gaps = 5/209 (2%)

XA_0051: 350453-350661= SEQ ID NO; 1

Cdsl 56 ESRKY-NFFIRTWTFVMISGFFITIASGHAWCIVLILGCQIATFKECIAVTSASGREK 114

+ KY + +IRT + M+ G F + GH + +++G 01 KE + + E+ XA 0051 350661 DK KYRSMWIRTCSSLWMLGGVFFIIYMGHLYIWAMWGIQIFMAKELFFLLRRAHEERR 350602

Cdsl 115 LPLTKTL WYLLFTTIYYLDGKSLFKFFQATF YEYPVL FIVTNHKFICYCLYLMG 170

LP + LNW+ FT + ++ G+ L + T + Y +++ ++ ICY LY+ G

XA 0051 350601 LPGFRLLNWHFFFTAMLFVYGRILQQQLVNTVSSDRFIYKLVSGLIKYQMVICYFLYIAG 350542

Cdsl 171 FVLFVCSLRKGFLKFQFGSLCOTHMVLLLWFQAHLIIKNVL GLFWFLLPCGLVIV DI 230

F+ F+ +L+K K+QFG TH +L++V Q+ + N+ G+FWFLLP L+ +ND+ XA 0051 350541 FMWILTLKKKMYKYQFGQYA THMILIVVFTQSSFTVANIFEGIFWFLLPAALIAM DV 350482 Cdsl 231 FAYLCGITFGKTKLIEISPKKTLEGFLGA 259

AY G FGKT LI++SPKKT EGF+GA

XA 0051 350481 AAYFFGFYFGKTPLIKLSPKKTWEGFIGA 350453

* Ckbl

A.SEQ ID NO: 2 Brassica sp. XA_0048r (Ckbl homologue)

SEVSGSDEEDLAWTTOFCKLPGNEFLCEVDDCFILDNFNLCGLRHQVPFYDNALDLI LDDDSSS >XA_0048r Length = 1689678

E-value = 1.15e-34, Score = 127, Bitscore = 53.5286,

Identities = 29/68 (42%), Positives = 38/68 (55%), Gaps = 4/68 (5%) XA_0048r: 346457-346520 = SEQ ID NO: 2

Ckbl 10 SR GSSDDEDSGAYDEW1PSFCSRFGHEYFCQVPTEFIEDDFNM SLSQEVPHYRKALDL 69

S SD+ED W FC G+E+ C+V FI D+FN+ L +VP Y ALDL

XA 0048r 346520 SEVSGSDEEDLA WTTWFC LPGNEFLCEVDDCFILDNFNLCGLRHQVPFYDNALDL 346465

Ckbl 70 ILDLEAMS 77

ILD ++ S

XA 0048r 346464 ILDDDSSS 346457

B. SEQ ID NO: 3 Brassica sp. XA_0048r (Ckbl homologue)

LIESAAEIJ-LYGLIHARYILTDKGFLSMLNKYNKSEFGRCPRVYCSGQSCLPIGLSDVP GASTVKIYCPKCEDIYH QPSKYQGSSSILLLVRIRYFSHLCVLVFFIHIKTDIDGSYFGTAFPHLFLMYYPSRRPKK VSSQSYVPRVFGFNL

H

>XA_0048r: Length = 1689678

E-value = 1.15e-34, Score = 281, Bitscore = 112.849,

Identities = 61/154 (39%), Positives = 88/154 (57%), Gaps 31/154 (20%)

XA 0048r: 346273-346423 = SEQ ID NO: 3 ,

54

Ckbl 118 IIEHAAEQLYGLIHARFILTKPGLQAMAEKFDHKEFGTCPRYYC GMQLLPCGLSDTVGK 177

+IE AAE LYGLIHAR+ILT G +M K++ EFG CPR YC+G LP GLSD G

XA 0048r 346423 LIESAAEMLYGLIHARYILTDKGFLSMLNKYNKSEFGRCPRVYCSGQSCLPIGLSDVPGA 346364

Ckbl 178 HTVRLYCPSCQDLYLPQSSRF LC LEGAFWGTS 209

TV++YCP C+D+Y Q S++ LC ++G+++GT+

XA 0048r 346363 STVKIYCPKCEDIY-HQPSKYQGSSSILLLVRIRYFSHLCVLVFFIHIKTDIDGSYFGTA 346305

Ckbl 210 FPGVFLKHFKELEEYVERKSKESYELKVFGFRIN 243

FP +FL ++ ++ S +SY +VFGF ++

XA 0048r 346304 FPHLFLMYYPSRRP—KKVSSQSYVPRVFGF LH 346273

* Rtc2

SEQ ID NO: 4 Brassica sp. XA_0011r (Rtc2 homologue)

GIVSLICWGVAEIPQIITNFRTKSSHGVSLSFLLAWVAGSVHSII

>XA_0011r Length ■ 4370687

E-value = 2.04e-04, Score = 105, Bitscore = 45.0542,

Identities = 18/45 (40%), Positives = 31/45 (68%), Gaps

(0%)

XA_0011r; 312819-312775 ■ SEQ ID NO: 4

Rtc2 18 GSISICC IWFVPQIYENFRRQSAEGLSLLFIVLWLLGDIFNVM 62

G +S+ CW V +PQI NFR +S+ G+SL F++ + G + +++

XA OOllr 312775 GIVSLICWGVAEIPQIITNFRTKSSHGVSLSFLLAWVAGSVHSII 312819 * SCB2

SEQ ID NO: 5 Brassica sp. XA_0001r (Scs2 homologue)

KVELKKQSSCSLQISNKTSTQWAFKVKTITiPRKYC PNTGVVLPGDSCNVTGDQLNCVFLFLFKNIYYTDQLN YVCLFLTLVTMQAQKEAPLDMQCKDKFLV >XA_0001r Length » 10813983

E-value = 2.04e-08, Score = 138, Bitscore = 57.7658,

Identities = 39/105 (37%), Positives = 50/105 (47%), Gaps = 31/105 (29%) XA._0001r; 1784455-1784559 =SEQ ID NO; 5

Scs2 14 KSPLTEQSTEYASISN-NSDQTIAFKVKTTAPKFYCVRPNAAWAPGE I QVQVI 67

K L +QS+ ISN S Q +AFKVKTT P+ YCVRPN W PG++ Q+ +

XA OOOlr 1784559 KWLKKQSSCSLQIS KTS QWAFKWITNPRKYCWPNTGVVLPGDSOTV GDQL CV 1784500 Scs2 68 FLGL TEEPAADFKCRDKFLV 87

FL L +E D +C+DKFLV

XA OOOlr 1784499 FLFLF*K IYYTDQL YVCLFLTLVTMQAQKEAPLI¾iQCKDKFLV 1784455

2. Ricinus sy Castor Bean ("Sbjct") vs S. cerevisiae matches ("Query") alignments:

* Cdsl

SEQ ID NO: 6 Ricinus sp. 29842 . m003508 (Cdsl homologue)

HRKRSNEWPEAAKENGGHLLVDDQNKYKSMWIRTCSTVWMIGS

RKAHEDRHLPGFRLLNVfflFFFTAMLFVYGRILSQRLVNTWSDKFLFQLWSLIKYH MAMCYFLYIAGFM FILT LKKK YKYQFGQYAWTHMILIVVFTQSSFTVANIFEGIFWFLLPASLIVINDIFAYICGFFFGRT PLIKLSPKKT WEGFIGASVTTMISAFVLANMMGRFQWLTCPRKDLSSGWLQCDPGPLFKPEYFILPFATV PQWFPWKEISILPVQW HALWLGLFASIIAPFGGFFASGFKRAFKIKDFGDSIPGHGGITDRMDCQMVMAVFAYIYH QSFWPQSISVEMIL DQILANL >29842.m003508 phosphatidate cytidylyltransferase, putative Length = 423

Score = 780 (279.6 bits), Expect = 1.4e-78, P = 1.4e-78

Identities = 152/385 (39%), Positives = 234/385 (60%) Sbjct; 18-399 ■ SEQ ID NO; 6

Query: 36 HKimSESWP-VTKESTA--A KESRKY- FFIRTVWTFWISGFFITLASGHAWCIVLI 91

H+ S V P KE+ + KY + +IRT T MI F + + GH + ++

Sbjct: 18 HRKRSNEWPEAAKENGGHIiLVDDQNKYKSIOTIRTCSTVWMIGSFALIVYMGHLYITAM V 77

Query: 92 I^CQIATFKECIAVTSASGREKi^PL KTLNWYLLFTTIYYLDGKSLFKFFQATF Y 147

+ QI KE + + +++LP + LNW+ FT + ++ G+ L + T +

Sbjct: 78 VVIQIYMAKELFNLLRKAHEDRHLPCFRLLNTCffl 137 Query: 148 EYPVLNFIVTNHKFICYCLYIJIGFVLFVCS 207

+ ++N ++ H +CY LY+ GF+ F+ +L+K K+QFG THM+L++V Q+ Sbjct: 138 LFQLV SLIKYHMAMCYFLYIAGFM FILTLKKKMYKYQFGQYAWTHMILIVVFTQSSFT 197 Query: 208 IKWLNGLFWFLLPCGLVIV DIFAYLCGITFGKTKLIEISPKKTLEGFLGAWFFTALAS 267

+ N+ G+FWFLLP L+++NDIFAY+CG FG+T LI++SPKKT EGF+GA T +++

Sbjct: 198 VA IFEGIFWFLLPASLIVI DIFAYICGFFFGRTPLIKLSPKK WEGFIGASVTTMISA 257 Query: 268 IILTRILSPYTYLTCPVEDLH OTFSNLTCEIJSiPVFLPQVYRLPPIFFDKVQINSITVKP 327

+L ++ + +LTCP +DL + + L C+ P+F P+ + LP I++ P

Sbjct: 258 FVIANM GRFQ LTCPRKDLSSGW LQCDPGPLFKPEYFILPEWVPQWFPWKEISILP 314

Query: 328 IYFHALNLATFASLFAPFGGFFASGLKRTFKVKDFGHSIPGHGGITDRVDCQFIMGSFA 387

+ +HAL L FAS+ APFGGFFASG KR FK+KDFG SIPGHGGITDR+DCQ +M FA Sbjct: 315 VQWHALWLGLFASIIAPFGGFFASGFKRAFKIKDFGDSIPGHGGI DRMDCQMVMAVFAY 374

Query: 388 LYYETFISEHRITVDTVLSTILMNL 412

+Y+++F+ I+V+ +L IL L

Sbjct: 375 IYHQSFWPQSISVEMILDQILA L 399 * Cho2

SEQ ID NO: 7 Ricinus sp. 30131.m007049 (C o2 homologue)

EKNLTKFFEEVNGIKTEMEEITNLLLDLQDLNEDSKSTHSTKVLKGIRDRINSD VTILRKAKIIKSRLESL

>30131.m007049 syntaxin, arabidopsis thaliana, putative, Length = 296

Score = 74 (31.1 bits), Expect = 3.8, P = 0.98

Identities = 21/73 (28%), Positives = 36/73 (49%)

Sbjct: 39-110 » SEQ ID NO: 7

Query: 796 EKDLTEFLTKV VLKDGKFRPLGNKFFG DSLKQLIK SIGVELSSEYMRRV GDAHVIS 855

EK+LT+F +VN +K + + N + L + K++ ++ R+N D I

Sbjct: 39 EK-^TKFFEFATOGIKT-I^EITNLLLDLQDLNEDSKSTHSTKVLKGIRDRINSDMVTIL 97 Query: 856 HRAWDIKQTLDSL 868

+A IK L+SL

Sbjct: 98 RKAKIIKSRLESL 110

* Ckbl

A. SEQ ID NO: 8 Ricinus sp. 29709.m001188 (Ckbl homologue) LVESAAE LYGLIHVRYILTSKGMSA LEKYK YDFGRCPRVYCCGQPCLPVGQSDIPRSSTVKIYCPKCEDIYY

PRSNVF

>29709.m001188 casein kinase II beta chain, putative

Length = 338

Score = 247 (92.0 bits), Expect = 1.6e-38, Sum P(2) = 1.6e-38 Identities = 43/81 (53%), Positives = 56/81 (69%)

Sbjct; 118-236 = SEQ ID NO: 8

Query: 118 IIEHAAEQLYGLIHARFILTKPGLQAMAEKFDHKEFG CPRYYCNGMQLLPGGLSDTVGK 177

++E AAE LYGLIH R+ILT G+ AM EK+ + +FG CPR YC G LP G SD Sbjct: 156 LVESAAEMLYGLIHVRYILTSKGMSAMLEKYKNYDFGRCPRVYCCGQPCLPVGQSDIPRS 215 Query: 178 HTVRLYCPSCQDLYLPQSSRF 198

TV++YCP C+D+Y P+S+ F

Sbjct: 216 STVKI CPKCEDI YPRSNVF 236

B.SEQ ID NO: 9 Ricinus sp. 29709.m001188 (Ckbl homologue)

DEESETDSEESDVSGSDGDiyrSWISWFCNLRGNEFFCEVDDEYIQDDFNLCGLSSQVPY YDYALDLILDVES

>29709.m001188 Score = 171 (65.3 bits), Expect = 1.6e-38, Sum P(2) = 1.6e-38

Identities = 33/72 (45%), Positives = 50/72 (69%)

Sbjct: 72-143 = SEQ ID NO: 9

Query: 7 EDYSRTGSSDDEDSGAYDE IPSFCSRFGHEYFCQVP EFIEDDFNMTSLSQEVPHY 63

++ S T S + + SG+ + WI FC+ G+E+FC+V E+I+DDF + LS +VP+Y

Sbjct: 72 DEESETDSEESDVSGSDGDDTSWISWFC LRGNEFFCEVDDEYIQDDF LCGLSSQVPYY 131

Query: 64 RKALDLILDLEA 75

ALDLILD+E+

Sbjct: 132 DYALDLILDVES 143

* Ino2

SEQ ID NO: 10 Ricinus sp. 29669.m000818 (Ino2 homologue)

HITPSMDSHLLANNRFIEEIPQSSWSSVPTQASEPDKTTDSEENSSQHQPLFKGQDH ₍

58

>29669.m000818 conserved hypothetical protein

Length = 87

Score = 62 (26.9 bits), Expect = 0.18, P = 0.16

Identities = 18/59 (30%), Positives = 24/59 (40%)

Sbjct: 27-83 = SEQ ID NO: 10

Query: 153 HIRSPKKQHRYTELNQRYPETHPHSNTGELPTNTA—DVPTEFTTREGPHQPI--GNDH 207

HI H N R+ E P S+ +PT + D . T+ HQP+ G DH Sbjct: 27 HITPSMDSHLIA-- NRFIEEIPQSSWSSVPTQASEPDKTTDSEENSSQHQPLFKGQDH 83

* Mrp835

SEQ ID NO: 11 Ricinus sp. 28355.m000102 (Mrps35 homologue)

PKLDEKVSCEYRELQEPVKIPGCVPIHGNKLLDPVQDRKNDAYKWFLHHSKRYKLADGIM VNSFTDLEGGAIKAL QEEEPAGKPPVYPVGPLVNMGSSSSREGAECLRWLDEQPHGSVLYVSFGSGGTLSYDQIN ELALGLEMSEQRFLW VARSPNDGVANA

>28355.m000102 UDP-glucosyltransferase, putative

Length = 426

Score = 79 (32.9 bits), Expect = 0.60, P = 0.45

Identities = 36/171 (21%), Positives = 75/171 (43%)

Sbjct: 132-293 = SEQ ID NO: 11

Query: 90 PDLERGQSLEHPVTKKPLQLRYDGTLGPPPVE KRLQNIFKDRLLQPFPSNPHCK NYVL 149

P L+ S E+ ++P+++ G P+ +L + +DR + H Y L

Sbjct: 132 PKLDEKVSCEYRELQEPVKIP GCVPIHGNKLLDPVQDRKNDAYKWFLHHSKRYKL 186

Query: 150 SPQLKQSIFEEITVEGLSAQQVSQKYGLKIPRVEAIVKLVSVENSW RRNRVSSDLKTMD 209

+ + + F ++ + A Q + G P V + LV++ +S +R + L+ +D

Sbjct: 187 AIX3IMVNSFTDLEGGAIKALQEEEPAGK--PPVYPVGPLV MGSSSSREG--AECLRWLD 242 Query: 210 ETLYR--MFPVFDSDASFKRENLSEIPVPQKTLASRFLTIAESEPFGPVDA 258

E + ++ F S + + ++E+ + + RFL +A S G +A

Sbjct: 243 EQPHGSVLWSFGSGGTLSYI¾INELALGLEMSEQRFLWVARSPNDGVANA 293

» Opx3 SEQ ID NO: 12 Ricinus sp. 30131.m006852 (Opi3 homologue)

LFGFGQFLNVRVYRLLGESGTYYGVRFGKSIP VTEFPFGVIRDPQYVGSILSLL

>30131.in006852 conserved hypothetical protein

Length = 164

Score = 103 (41.3 bits), Expect = 3.6e-05, P = 3.6

Identities = 27/56 (48%), Positives = 31/56 (55%)

Sbjct; 72-126 = SEQ ID NO: 12

Query: 104 LFGLGQVLVLSSMYKLGITGTYLGDYFGIIJmERVTGFPF VSN PMYQGSTLSFL 159

LFG GQ L + LG +GTY G FG + VT FPF V +P Y GS LS L

Sbjct: 72 LFGFGQFLNVRVYRLLGESGTYYGVRFGKSIPW-VTEFPFGVIRDPQYVGSILSLL 126

* Rtc2

A. SEQ ID NO: 13 Ricinus sp. 29250.m000234 (Rtc2 homologue)

GFVSLVSWGVAEVPQIITNFRTKSSHGVSLLFLLTWVAGDVFNLVGCLLEPATLPTQFYT ALLYTTSTIVLVLQG LYYD

>29250.m000234 conserved hypothetical protein

Length = 377

Score = 184 (69.8 bits), Expect = 2.8e-25, Sum P(2) = 2.8e-25 Identities = 33/79 (41%), Positives = 54/79 (68%)

Sbjct: 37-115 = SEQ ID NO: 13

Query: 18 GSISICCWIWFVPQIYE FRRQSAEGLSLLFIVL LLGDIF VMGAMMQ L-LPTMIIL 76

G +S+ W V VPQI NFR +S+ G+SLLF++ W+ GD+FN++G +++ LPT Sbjct: 37 GFVSLVS GVAEVPQIITNFRTKSSHGVSLLFLLTWVAGDVFNLVGCLLEPATLPTQFYT 96 Query: 77 AAYYTLADLILLIQCMWYD 95

A YT + ++L++Q ++YD

Sbjct: 97 ALLYTTSTIVLVLQGLYYD 115

B. SEQ ID NO: 14 Ricinus sp. 29250.m000234 (Rtc2 homologue)

QWLGWLMAAIYMGGRIPQIWLNIKRGSVEGLNPLMFIFALVA^

LDFFIILQYVYY p

60 conserved hypothetical protein

Score = 150 (57.9 bits), Expect = 2.8e-25, Sum P(2) = 2.8e-25 Identities = 32/87 (36%), Positives = 53/87 (60%)

Sbjct: 269-355 = SEQ ID NO: 14

Query: 209 QILGYLSAILYLGSRIPQIVL FKRKSCEGVSFLFFLFACLGNTSFIISVL 259

Q LG+li A +Y+G RIPQI LN KR S EG++ L F+FA + N ++++S++

Sbjct: 269 QWLGWLMAAI MGGRIPQIWLNIKRGSVEGLNPL FIFALVANLTYVLSIWR TEWESI 328 Query: 260 --SASWLIGSAGTLLMDFTVFIQFFLY 284

+ WL+ +A + +DF + +Q+ Y

Sbjct: 329 KANMPWLLDAAVCVALDFFIILQYVYY 355

C. SEQ ID NO: 15 Ricinus sp. 29250.m000234 (Rtc2 homologue)

GFVSLVSWGVAWPQIITNFRTKSSHGVSLLFLLT AGDVFNLVGCL conserved hypothetical protein

Score = 83 (34.3 bits), Expect = 0.14, P = 0.13

Identities = 17/48 (35%), Positives = 28/48 (58%)

Sbjct: 37-84 ■ SEQ ID NO: 15

Query: 212 GYLSAILYLGSRIPQIVLNFKRKSCEGVSFLFFLFACLGNTSFIISVL 259

G++S + + + +PQI+ F+ KS GVS LF L G+ ++ L

Sbjct: 37 GFVSLVSWGVAEVPQII NFRTKSSHGVSLLFLLTWVAGDVFNLVGCL 84

D. SEQ ID NO: 16 Ricinus sp. 29250.m000234 (Rtc2 homologue)

IPQIWLNIKRGSVEGLNPLMFIFALVANLTYVLSIWRTTEWESIKANMPWLLDAAVCVAL DFFIILQYVYYRYF REKK conserved hypothetical protein

Score = 72 (30.4 bits), Expect = 2.4, P = 0.91

Identities = 21/79 (26%), Positives = 43/79 (54%)

Sbjct: 284-362 = SEQ ID NO: 16 .

61

30 VPQIYENFRRQSAEGLSLLFIVLWLLGDIF VMGAMM Q LLPTM--IILAAYYTL 82

+PQI+ N +R S EGL+ L + L+ ++ V+ ++ +++ M ++ AA

284 IPQIWLNIKRGSVEGL PLMFIFALVANLTYVLSIVVR TEWESIKANMPWLLDAAVCVA 343 83 ADLILLIQCMWYD--KEKK 99

D +++Q ++Y +EKK

344 LDFFIILQYVYYRYFREKK 362

*Scs2

SEQ ID NO: 17 Kicinus sp. 30174.m008835 (Scs2 homologue)

LNIQPSELKFPFELKKQSSCSMQLTl^SNSWAFKVKTTNPKKYCVRPNTGIILPGTA CNV V M

QCKDKFLLQSVAAPISVTTKDITADTFTKEDGKVIEEFKLRVVYIPANPPSPVPEEPEEG LSPRSSVLENGDQDS SLLEAVSRSLEEPKE

>30174.m008835 vesicle-associated membrane protein, putative Length = 238

Score = 200 (75.5 bits), Expect = 4.0e-17, P = 4.0e-17

Identities = 55/171 (32%), Positives = 85/171 (49%)

Sbjcti 7-171 ■ SEQ ID NO: 17

Query: 4 VEISPDVLVYKSPLTEQSTEYASISN SDQTIAFKVKTTAPKFYCVRPNAAWAPGETIQ 63

+ I P L + L +QS+ ++N S+ +AFKVKTT PK YCVRPN ++ PG Sbjct: 7 LNIQPSELKFPFFiKKQSSCSMQLT^SNSYVAFKVKTTNPKKYCVRPNTGIILPGTACN 66

Query: 64 VQVIFLGLTEEPAADFKCRDKFLVITLPSPYDL GKAVADVWSDLEAEFKQQAISK-KIK 122

V V E P D +C+DKFL+ ++ +P +G D+ +D + + I + K++

Sbjct: 67 V V MQAQKEAPP-DMQCKDKFLLQSVAAP DGVTTKDITADTFTKEDGKVIEEFKLR 122

Query: 123 VKYLIS--PDVHPAQ-NQNIQENKETVEPWQDSEPKEVPAWNEKEVPAE 170

Y+ + P P + + + +E QDS E AV E P E

Sbjct: 123 WYIPANPPSPVPEEPEEGLSPRSSVLENGDQDSSLLE--AVSRSLEEPKE 171

Glycine spJsoybean ("Sbict") vs S. cerevisiae ("Query") alignments: * CdBl

From Phytozome:

HSP#1 Sxibject 41076229-41076035=SEQ ID NO

SEQ ID NO: 18 Glycine sp. (Cdsl homologue) PIIPGTSE HNLRPIICCLMQFS KEISILPIQWHSLCLGLFASIIAPFGGFFASGFKRAFKIK

HSP#2 Subject 41075939-41075877=SEQ ID NO: 19

SEQ ID NO: 19 Glycine sp. (Cdsl homologue)

LQDFGDSIPGHGGITDRMDCQ

HSP#3 Subject 41077510-41077325-SEQ ID NO: 20

SEQ ID NO: 20 Glycine sp. (Cdsl homologue)

LDLLYRFLLPATLIVINDIAAYIFGFFFGRTPLIKLSPKKTWEGFIGASVTTIISAFMVS RI

HSP#4 Subject 41078414-41078229=SEQ ID NO: 21

SEQ ID NO: 21 Glycine sp. (Cdsl homologue)

CYCFGYLPFYWLSAGFMWFILTLKKKMYKYQFGQYAWTHMILIWFGQSSFTVASIFEGIF W

Defllne Score E Query View

457

Gm10 54.7 2.36-13 301-360

359-379 212-273 163-217 open all : close all

E Feature #1

B HSF#1 : Score: 54.7 bits (130.0) E-value: 2.3e-13 Identity: 46.2% (30/65) Positive: 64.6% (42/65) Frame: +0/-1

Subject I 41076229-41076035 - SEQ ID NO: 18

Query 301 PVFLP QVYRLPPIFFDKVQIN—3ITVKPIYFHALNLATFA3LFAPFGGFFASGIKRTFKVK36 0

P+ +P + + L PI +Q + I++ PI +H+L L FAS+ APFGGFFASG KR F +K

Subject 41076229PI*IPGSENHLRPIICCLMQFSeKEISILPIQBHSLCLGLFASIIAPFGGFF ASGFKRAFIK41076035

B HSP#2: Score: 43.9 bits (102.0) E-value: 2.3e-13 Identity: 81.0% (17/21 ) Positive: 95.2% (20/21 ) Frame: +0/-3

Subject 41075939-41075877 - SEQ IP N i: 19

Query 359VKDFGHSIPGHGGITDRVDCQ379

++DFG SIPGHGGITDR+DCQ

Subject 41075939LQDFGDSIPGHGGITDRMDCQ41075877

B HSP#3: Score: 64.7 bits (156.0) E-value: 4.5e-9 Identity: 50.0% (31/62) Positive: 75.8% (47/62) Frame: +0/-

Subject 41077510-41077325 - SEQ ID NO: 20

Query 212L GLFWFLLPCGLVIVN0IFAYLCGI FGKTKLIEISPKKTLEGFLGAKFFTALA3IIL RI273

1+ L+ FLLP L+++NDI AY+ G FG+T LI++SPKKT EGF+GA T +++ +++RI

Subject 41077510LDLLYRFLLPATLIVINDIAAYIFGFFFGRTPLIKLSPKKTIIEGFIGASVT TIISAFMVSRI41077325

37.1% (23/62) Positive: 58.1% (36/62) Frame: +0/-3

Query 163CYCL-YL MGFVLFVCSLRKGFLKFQFGSLCVTHMVILLWFQAHLIIKNVLNGLFB217

CYC YL GF+ F+ +L+K K+QFG THM+1++V Q+ + ++ G+FW

Subject 41078414CYCFGYLPFYILSAGFMBFILllKKKMYKYQFGQYAITHMILIWFGQSSFTV ASIFEGIFB41078229 * Ckbl

From Phytozome:

Feature#l/HSP#l Subject 5095580-5095903=SEQ ID NO: 22

SEQ ID NO: 22 Glycine sp. (Ckbl homologue)

LIESAAEMLYGLIHARYVLTSKGMAAMVSFLLVDHSFCSLSSWRLALLVTSLELLQL D YKNYDFGRCPRVYCSG QPCLPVGQSDIPRSSTVKIYCPRCEDLYYPRS

Feature#l/HSP#3 Subject 5095083-5095259=SEQ ID NO: 23

SEQ ID NO: 23 Glycine sp. (Ckbl homologue)

SGSDGDDTSWISWFCNLRGNEFFCEVDDDYIQDDFNLCGLSSQVPYYDYALDLILDV ES

Feature#2/HSP#2 Subject 5060232-5060552=SEQ ID NO: 24

SEQ ID NO: 24 Glycine sp. (Ckbl homologue)

LIESAAI^YGLIHARYILTSKGMAAMVFFSLLSCFLILYCGQNWHLMTLDYLQLHKYKNY NFGRCPRVFCSGQP CLPVGQSDVPRSSTVKIYCPRCE

Feature#2/HSP#4 Subject 5059804-5059953=SEQ ID NO: 25

SEQ ID NO: 25 Glycine sp. (Ckbl homologue)

WISWFCNLRGNEFFCEVDDDFVQDDFNLCGLSSQVPYYDYALDLILDVES

,

64

Defline Score E Query View

■ Gm05 82.4 9e-15 ^BOtsaat*

JSmBKSBHK* open all : close all

a Feature #1

S HSP#1 : Score. 82.4 bits (202.0) E-value: 9e-15 Identity 39.8% (43/108) Positive: 51.9% (56/108) Frame: +0/+2

Subject 5095580-5095903 " SEQ ID MO: 22

Query 118 lIEHmOMGIIHARriLTKPGIiQAMA ^< · EmHKEEGTCPRnCB(»)a.lfCGlSPrwGKHIVKLKPSC l87

+IE AAE IiYGIIHAR++IiT G+ AM +K+ + +FG CPR YC+G LP G 3D TV-+YCP C

Subject 5M5580LIESAAEffiYGLI8AR-VLTSKGMAAMYSFIIYD*HSF^

Query 188 195

Subject 5095880 5095903

S HSP#3; Score: 65.1 bits ( 157 0) E-value: 1.3Θ-9 Identity: 48.4% (31/64) Positive: 70 3% (45/64) Frame: +0/+3

Subject 5095083-5095259 - SEQ IP NO: 23

Query 12 TGS3DDEDSGAYDEWIP5FCSRrGHEYFCCVPTEF HDLEA75

+GS D+ S G+E+FC+V ++I LS +VP+Y +E+

Subject 5095083 SGSDGDDTS 3 3 5095259

i Feature #2

3 HSP#2: Score: 80.1 bits (196.0) E-value: 4.9e-14 Identity: 39.3% (42/107) Positive: 50.5% (54/107) Frame: +0/+3

Subject 5060232-5060552 - SEQ IP NOi 24

Query 118 10 EKFDHKEFGTCPRYYCHGMCMPCGIiSDTVGKHTVRIiYCPSCQ 188

+IE AAE IiYGLIHAR+Π,Τ AM K+ + FG CPR +G G TV++YCP C+

Subject 5060232lIESAAEMLY<aiHARYILTSKGMAAMV*FFSIiSCFLILYC^

Query 189 195

+Y p+s

Subject 5060532 5060552

3 HSP#4: Score: 49.7 bits (117,0) E-value: 6.5e-5 Identity: 54.0% (27/50) Positive: 78.0% (39/50) Frame- +07+ 1

Subject 5059804-5059953 - SEQ IP NO 25

Query 26WIPSrCSRFGHEIFCQVPlEFIEDDFHMTSIi3QmHYRI AIDin,DIEA75

«I FC+ G+E+FC+V +F++DDFI+ IS +VP+Y ALDLHD+E+

Subject 5059804WISWCKI.RGIIEFrCEVDDrFVCjDDFII.C5LSSCWYYDYAI,DlIIDVES 5059953

From NCBI:

SEQ ID NO: 26 Glycine sp. ACU23831.1 (Ckbl homologue)

SGSDGDDTSWISWFCNLRGNEFFCEVDGDYIQDDFNLCGLSSQVPYYDYALDLILDV ESSHGDMFTEEQNELIES AAEMLYGLIHTRYVLTSKGMAAl^DKYKNYDFGRCPRVYCSGQPCLPVGQSDIPRSSTVK IYCPRCEDLYYPRSK YQGNIDGAYFGTTFPHLFLMTYGQLKPQKPAQGYVPRVFGFKVH

>gb|ACU23831.1 | unknown [Glycine max]

Length=261

Score = 171 bits (434), Expect = 8e-44, Method:

Compositional matrix adjust.

Identities = 92/236 (39%), Positives = 136/236 (58%), Gaps =

46/236 (19%) ,

65

Sbjct: 66-259 = SEQ ID NO: 26

Query 12 TGSSDDEDSGAYDEWIPSFCSRFGHEYFCQVPTEFIEDDFNM SLSQEVPHYRKALDLIL 71

+GS D+ S WI FC+ G+E+FC+V ++I+DDFN+ LS +VP+Y ALDLlL

Sbjct 66 SGSDGDDTS WISWFC LRGNEFFCEVDGDYIQDDFNLCGLSSQVPYYDYALDLIL 120 Query 72 DLEA MSDEEEDEDDWEEDEVDQEMQSNDGHDEGKRRNKSPWNKS11EHAAEQLY 127

D+E+ M EE++E +IE AAE LY

Sbjct 121 DVESSHGDMFTEEQNE LIESAAEMLY 146

Query 128 GLIHARFILTKPGLQAMAEKFDHKEFG CPRYYCNGMQLLPCGLSDTVGKH VRLYCPSC 187

GLIH R++LT G+ AM +K+ + +FG CPR YC+G LP G SD TV++YCP C Sbjct 147 GLIHTRYVLTSKGMAAMLDKYKNYDFGRCPRVYCSGQPCLPVGQSDIPRSSTVKIYCPRC 206 Query 188 QDLYLPQSSRFLCLEGAFWGTSFPGVFLKHFKELEEYVERKSKESYELKVFGFRIN 243

+DLY P+S ++GA++GT+FP +FL + +L+ +K + Y +VFGF+++

Sbjct 207 EDLYYPRSKYQGNIDGAYFGTTFPHLFLMTYGQLK PQKPAQGYVPRVFGFKVH 259 Prodom search prediction

SEQ ID NO: 27 ^* Glycine sp. PD003829 (KINASE II BETA CASEIN SUBUNIT CK PHOSPHORYLATION PATHWAY SIGNALING WNT)

ISWFCNLRGNEFFCEVDDEYIQDDFNLCGLSSQVPYYDYALDLILDVESSHGDMFTEEQN ELVESAAEMLYGLIH YILTSKGMAAMLEKYKNYDFGRCPRVYCCGQPCLPVGQSDIPRSSTVKIYCPKCEDIYYP RSKYQGNIDGAYF GTTFPHLFLMTYGHLKPQKSTQNYVPRVFGFKLH

>PD003829 (Closest domain: Q8LPD2_TOBAC 95-278)

Number of domains in family: 137

Commentary (automatic) :

KINASE II BETA CASEIN SUBUNIT CK PHOSPHORYLATION PATHWAY SIGNALING WNT

Length = 184

Score = 940 (366.7 bits), Expect = 6e-101

Identities = 170/184 (92%), Positives = 177/184 (96%)

Sbjct; 95-278 = SEQ ID NO; 27

Query: 76 ISWFCNLRGNEFFCEVDGDYIQDDF LCGLSSQVPYYDYALDLILDVESSHGDMFTEEQN 135

ISWFC LRGNEFFCEVD +YIQDDFNLCGLSSQVPYYDYALDLILDVESSHGDMFTEEQN Sbjct: 95 ISWFCNLRGNEFFCEVDDEYIQDDFNLCGLSSQVPYYDYALDLILDVESSHGDMFTEEQN 154

Query: 136 ELIESAAEMLYGLIHTRYVLTSKGMAAMLDKYK YDFGRCPRVYCSGQPCLPVGQSDIPR 195

EL+ESAAEMLYGLIH RY+LTSKGMAAML+KYKNYDFGRCPRVYC GQPCLPVGQSDIPR Sbjct: 155 ELVESAAEMLYGLIHVRYILTSKGMAAMLEKYK YDFGRCPRVYCCGQPCLPVGQSDIPR 214 Query: 196 SS VKIYCPRCEDLYYPRSKYQGNIDGAYFG TFPHLFLM YGQLKPQKPAQGYVPRVFG 255

SSTVKIYCP+CED+YYPRSKYQGNIDGAYFGT FPHLFLMTYG LKPQK Q YVPRVFG Sbjct: 215 SSTVKIYCPKCEDIYYPRSKYQGNIDGAYFGTTFPHLFLMTYGHLKPQKSTQ YVPRVFG 274 Query: 256 FKVH 259

FK+H

Sbjct: 275 FKLH 278

NCBI :

SEQ ID NO: 28 ^* Glycine sp. ACO91805.1 (phospholipid N methyltransferase) PLIAFGQFLNFRVYQLLGETGTYYGVRFGKTI-PWVTEFPFGVIKDPQYVGSIMSILA

>gb[ ACO91805.1 | phospholipid N-methyltransferase [Glycine max] GENE ID: 100301902 PLMT | phospholipid N-methyltransferase [Glycine max

(10 or fewer PubMed links)

Score = 38.1 bits (87), Expect = 8e-04, Method: Compositional matrix adjust.

Identities = 25/58 (43%), Positives = 28/58 (48%), Gaps = 1/58 (2%) Sbjct: 74-130 = SEQ ID NO; 28

Query 103 ALFGLGQVLVLSSMYKIJGITG YIJGDYFGIIJ DERVTGFPFWSN PMYQGSTLSFIXS 160

L GQ L LG GTY G FG + VT FPF V +Ρ Y GS +S L

Sbjct 74 PLIAFGQFL FRVYQLLGETGTYYGVRFGKTI-PWV EFPFGVIKDPQYVGSIMSILA 130 * Rtc2

Phytozome :

Gm03 subject 35049077-35048961=SEQ ID NO: 29

SEQ ID NO: 29 Glycine sp. (Rtc2 homologue)

GLISVIVWWAEIPQILTNYRTKSAEGLSVTFLITWIIG .

67

Gml9 subject 37947353-37947237=SEQ ID NO: 30

SEQ ID NO: 30 Glycine sp. (Rtc2 homologue)

GLI VIVWWAEIPQIIPNYRTKSAEGLSVTFLV WIIG

Gmll subject 4783865-4784035«SEQ ID NO: 31

SEQ ID NO: 31 Glycine sp. (Rtc2 homologue)

GFISLICWGVAEIPQII NFRAKSSHGVSLAFLL WVAGLVSLSTFLHFLIVLSKSY

GmOl subject 50607082-50606966=SEQ ID NO: 32

SEQ ID NO: 32 Glycine sp. (Rtc2 homologue)

GFISLVCWGVAEIPQIITNFRAKSSHGVSLAFLLTWVAG

Defline Score E QueryView

■ HI hill HI 11 29¾

Gm03 47.8 2.50e-4 < - s Feature #1 subject 350 077-350 8 61- SEQ ID MO; 29

8HSP#1: Score: 47.8 bits (112.0) E-value: 2.50e-4 Identity: 48.7% (19/39) Positive: 74.4% (29/39) Frame: +0/-3

Query 18GSI3ICCIIWFVPQIYENFRR¾SAEGLSLLFIVLBLLG56

G IS+ I+V +PQI N+R +SAEGLS+ F++ B++G

Subject 35049077GLISVr/B AEIP5ILTNYRTKSAEGL5VTFLITHIIG35048961

Gm19 46.2 6.76e ^{®¾ 18} " ⁵⁶

9 Feature #1 subject 37947353-37947237 - SEQ ID MO: 30

fe! HSP#1: Score: 46.2 bits (108.0) E-value: 6.76e-4 Identity: 48.7% (19/39) Positive: 74.4% (29/39) Frame: +0/-2

Query 18GSISICCIIWFVPQIYENFRRQSAEGISLLFIVL»LLG56

G I++ I+V +PQI N+R +SAEGLS+ F+V B++G

Subject 37947353GLIWIVlinA¾EIPQIIPNYRTOSftEGLSTOLVTBnG37947237

Gm11 45.1 1.95e-3 i i ^' - 18-8Ο

Feature #1 Subject 4783865-4784035 - SEQ ID MO: 31

BHSP#1: Score: 45.1 bits (105.0) E-value: 1.95e-3 Identity: 36.5% (23/63) Positive: 57.1% (36/63) Frame: +0/+2

Query 18GSISICCBIVVFVPQIYENFRRQSAEGLSLLFWLHLLGDIFNVMGAMMQNLLPIMIIL YY80

G IS+ CB V +PQI NFR +S+ G+SL F++ 1+ G + + L +I+L+ Y

Subject 4783865 GFISLICBGVAEIPQIITNFRAKSSHGVSLAFLLTBVAGLV- SLSTFLHf LIVLS SY478 035

Gm01 44.7 2.30e-3 ⁶

Feature #1 subjec 50607082-506O6 66 - SEQ ID O: 32

BHSP#1: Score: 44.7 bits (104.0) E-value: 2.30e-3 Identity: 48.7% (19/39) Positive: 69.2% (27/39) Frame: +0/-2

Query 18GSI3ICCBIWFVPQIYENFRRQSAEGLSLLFIVL»LLG56

G 15+ CB V +PQI NFR +S+ G+SL F++ »+ G

Subject 50607082 GF I S LVCBGVAE I PQI ITNFRAK S S HGVS LAFLLTWVAG 50606966

NCBI :

gbj ACU24122.1 | unknown [Glycine max]

Length=379

A. SEQ ID NO: 33 Glycine sp. ACU24122.1 (Rtc2 homologue)

GLTSLVFWGVAEIPQIITIFRTKKSHGVSLVFLLTWVAGDICNLTGCILEPATLPTQ YYT ALLYTITTIVLLLLIVYYDYISRWYKHRQKVNLVRDH ,

69

Score = 57.8 bits (138), Expect = 2e-09, Method:

Compositional matrix adjust.

Identities = 32/97 (33%), Positives = 53/97 (55%), Gaps = 1/97 (1%)

Sbjct: 37-133 = SEQ ID NO: 33

Query 18 GSISICCTI FVPQIYENFRRQSAECLSLLFIVLWLLGDIF VMGAMMQ-NLLPTMIIL 76

G S+ W V +PQI FR + + G+SL+F++ W+ GDI N+ G +++ LPT

Sbjct 37 GLTSLVFWGVAEIPQIITIFRTKKSHGVSLVFLLTWVAGDICNLTGCILEPATLPTQYYT 96 Query 77 AAYYTLADLILLIQ^MWYDKEKKSIIjQEVKKNVDFVH 113

A YT+ ++LL+ ++YD + K N+ H

Sbjct 97 ALLYTITTIVLLLLIVYYDYISRWYKHRQKV LVRDH 133

B. SEQ ID NO: 34 Glycine sp. ACU24122.1 (Rtc2 homologue)

YEKHSTFGQWLGWLMAAIYISGRVPQIWLNIKRSSVEGLNPFMFVFALVANVTYVGS IL

Score = 53.1 bits (126), Expect = 5e-08, Method: Compositional matrix adjust.

Identities = 24/59 (41%), Positives = 39/59 (66%), Gaps = 0/59 (0%)

Sbjct t 264-322 ■ SEQ ID NOt 34

Query 201 FEQINLPAQILGYLSAILYLGSRIPQIVLNFKRKSCEGVSFLFFLFACLGNTSFIISVL 259

+E+ + Q LG+L A +Y+ R+PQI LN KR S EG++ F+FA + N +++ S+L Sbjct 264 YEKHSTFGQWLGWLMAAIYISGRVPQIWLNIKRSSVEGLNPFMFVFALVANVTYVGSIL 322-

C. SEQ ID NO: 35 Glycine sp. ACU24122.1 (Rtc2 homologue)

LTSLVFWGVAEIPQIITIFRTKKSHGVSLVFLLTWVAGD Score = 28.9 bits (63), Expect = 0.85, Method: Compositional matrix adjust.

Identities = 14/39 (36%), Positives = 24/39 (62%), Gaps = 1/39 (3%)

Sbjct: 38-76 - SEQ ID NO; 35 Query 214 LSAILYLG-SRIPQIVLNFKRKSCEGVSFLFFLFACLGN 251

L+++++ G + IPQI+ F+ K GVS +F L G+

Sbjct 38 LTSLVFWGVAEIPQII IFRTKKSHGVSLVFLLTWVAGD 76 D.SEQ ID NO: 36 Glycine sp. ACU24122.1 (Rtc2 homologue)

VPQIWLNIKRSSVEGLNPFMFVFALVANV

Score = 27.7 bits (60), Expect = 2.1, Method: Compositional matrix adjust.

Identities = 12/29 (41%), Positives = 18/29 (62%), Gaps = 0/29 (0%)

Sbjct I 287-315..» SEQ ID NO; 36

Query 30 VPQIYENFRRQSAEGLSLLFIVLWLLGDI 58

VPQI+ N +R S EGL+ V L+ ++

Sbjct 287 VPQIWLNIKRSSVEGLNPFMFVFALVANV 315

* Scs2

GmOl Subject 44980635-44980751 = SEQ ID NO: 37

SEQ ID NO: 37 Glycine sp. (Scs2 homologue)

QFMVLQVKTTNPKKYCVRPNTGVVMPRSTCDVIGFFFSL

Gm05 Subject 3726981-3726889 = SEQ ID NO: 38

SEQ ID NO: 38 Glycine sp. (Scs2 homologue)

CLFFKVKT NPKKYCVRPNTGIVTPRSTCDV

Gm03 Subject 3637572-3637432 = SEQ ID NO: 39

SEQ ID NO: 39 Glycine sp. (Scs2 homologue)

TCNFDLGVHFMVLQVKTTNPKKYCVRPNTGWMPRSTCDVIGFFFSL

Phytozome : .

71

Defllne Score Query View

mm mnmrmm it

Gm01 40.8 2.64e-2

Feature #1 Subject 44980635-44980751 = SEQ ID NO; 37

E HSP#1 : Score: 40.8 bits (94.0) E-value: 2.64e-2 Identity: 51.3% (20/39) Positive: 56.4% (22/39) Frame: +0/+3

Query 33QTIAFVKTTAPKFYCVRPHRAWAPGETI¾V¾VIFLGL71

¾ + +VKTT PK YCVRPN W P T V F L

Subject 44980635QFM\^QVKTT PKKYCVRPNTGVVMPR3TCDVIGFFF3L4980751

Gm05 39.7 5.10e-2 *>·«

Feature #1 ^Sub J ^act 3726981-3726889 - SEQ ID NO: 38

B HSP#1 : Score: 39.7 bits (91.0) E-value: 5.10e-2 Identity: 58.1% (18/31 ) Positive: 64.5% (20/31 ) Frame: +0/-3

Query 34TIAFKVKTTAPKFYCTRPNAAVVAPGETIQV64

+ FKVKTT PK YCVRPN +V P T V

Subject 3726981 CLFFVKTTNPKKYCVRPNTGIVTPRSTCDV3726889

Gm03 39.7 5.10e^2 28

Feature #1 ^Sub ^* ^ct 3637572-3637432 - SEQ ID NO: 39

B HSP#1 : Score: 39.7 bits (91.0) E-value: 5.1 Oe-2 Identity: 46.8% (22/47) Positive: 53.2% (25/47) Frame: +0/-2

Query 28SNNSDQ IAF—KVKTTAP FYCVRPNAAWAPGETIQVQVIFLGL?!

+ N D + F +VKTT PK YCVRPN W P T V F L

Subject 3637572TCNFDLGVHFMVLQVKTTNPKKYCVRPNGVVMPRS CDVIGFFFSL3637432

NCBI:

SEQ ID NO: 40 Glycine sp. ACU21258.1 (Scs2 homologue)

LHIEPAELRFVFELKKQSSCLVQLAI^TDHFLAFKWTTSPKKYCWPNIGIIKPNDKC DFTVTMQAQRMAPPDM LCKDKFLIQSTWPVGTTEDDITSDMFAKDSGKFIEEKKLRWLISP

>gb| ACU21258.1 | unknown [Glycine max]

Length=295

Score = 73.6 bits (179), Expect = 2e-14, Method:

Compositional matrix adjust.

Identities = 47/127 (37%), Positives = 68/127 (54%), Gaps =

6/127 (5%) Sbjct: 6-127 = SEQ ID NO: 40

Query 4 VEISPDVLVYKSPLTEQSTEYASIS NSDQ IAFKVKTTAPKFYCVRPNAAWAPGETIQ 63

+ I P L + L +QS+ ++N +D +AFKVKTT+PK YCVRPN ++ P +

Sbjct 6 LHIEPAELRFVFELKKQSSCLVQl-ANNTDHFLAFKVKTTSPKKYCVRPNIGIIKPNDKC D 65

Query 64 VQVIFLGLTEEPAADFKCRDKFLVITLPSPYDLNGKAVADVWSDLEAEFKQQAI-SKKIK 122

V P D C+DKFL+ + P G D+ SD+ A+ + 1 KK++

Sbjct 66 FTV MQAQRMAP-PDMLCKDKFLIQSTWPV GT EDDI SDMFAKDSGKFIEEKKLR 121 .

72

Query 123 VKYLISP 129

V LISP

Sbjct 122 V-VLISP 127

Example 4: knockdown of gene homologues in Arabidopsis thaliana

As noted in the Examples above, genes were identified in Saccharomyces and Chlamydomonas which, when knocked down, were observed to increase lipid droplet accumulation and the production of triacyl glycerols with saturated fatty acid chains. Homologues of these genes were identified in the plant Arabidopsis (a model plant and relative of the Brassicas), and mutant Arabidopsis plants were generated in which at least one of the identified gene homologues was knocked down.

Arabidopsis mutants were obtained from a commercial service (NASC - The European Arabidopsis Stock Centre). The Arabidopsis mutants were generated by T-DNA Insertional Mutagenesis, and a description of this methodology is provided in O'Malley and Ecker, (2010), "Linking genotype to phenotype using the Arabidopsis unimutant collection", The Plant Journal, 61, 928-940 (the contents of which are incorporated herein by reference in their entirety).

The Arabidopsis mutants are single, segregating flank-tagged T3-generation transferred DNA (T-DNA) lines and were generated by vacuum infiltration of Columbia (Col) plants (Columbia-0; CS60000, the sequenced genome) with Agrobacterium tumefaciens vector pROK2, a derivative of the pBIN19 vector (see Baulcombe et al. (1986), "Expression of biologically-active viral satellite RNA from the nuclear genome of transformed plants", Nature, 321 (6068):446-449).

T-DNA transformed plants were grown, genomic DNA prepared, and T-DNA flanking plant DNA was recovered and sequenced. Insertion site sequences were aligned with the Arabidopsis genome sequence and gene annotation added. Kanamycin (ΝΤΡΠ marker) was employed for selection of plants having T-DNA insertions. The presence of the T-DNA insertion in the gene was determined by PCR using primers from the T-DNA Left-border and the target gene sequence.

For PCR 1, the LBal primer was used: 5' tggttcacgtagtgggccatcg 3' (SEQ ID NO: 42). For PCR 2 and sequencing, the LBbl primer was used: 5' gcgtggaccgcttgctgcaact 3' (SEQ ID NO: 43). Separate Arabidopsis mutants were generated each having one of the following genes knocked out by T-DNA insertion: CDS1, CDS2, RTC2, C bl or SCS2, a shown in Table 2 below. Table 2

TAIR search results

Gene Match to

ID Brassica Score Allele

Phenoty Seed

Best hits (locus name) E Germpiasm name (other name) Mutage Genotype

pe population value n

AT1G62430 T-DNA

SALK_130379(N630379) heterozygous NA T2 and T3 insertion

CDSl. CDP-

6e-83

DIACYLGLYCEROL SYNTHASE 1

Not found in NCBI SALK_088268C(N657989) = homozygous NA NA

S ALK_079137(N579137) = heterozygous NA T2 and T3

XA_0051

Length =

1633677 SALK_148049(N648049) = heterozygous NA T2 and T3

CDS1 E-value = AT4G22340

1.37e-34 CDS2,

CYTIDINEDIPHOSPH ATE 5e-82 SALK_148057(N648057) = heterozygous NA T2 and T3

D1ACYLGLYCEROL

SYNTHASE 2

found in NCBI

SAJLK_099882C(N68151 ) = homozygous NA NA

SALK_106246C(N670013) = homozygous NA NA

XA_0011r

AT4G20100 SALK_128175(N628175) = NA NA T2 and T3 Length =

RTC2 PQ-loop repeat family

4370687 protein / transmembrane le-26

E-value =

family protein

2.04e-04

found in NCBI SALK_108796C (N682757) = homozygous NA NA

SALKJ40678 (N640678) = heterozygous NA T2 and T3

XA_0048r

Length =

1689678

E-value =

AT5G47080 SALK_140689 (N640689) = heterozygous NA T2 and T3 1.15e-34

CKB1 CASEIN

&

CKbl KINASE II BETA 3e-42

XA_0048r CHAIN 1

Length = found in NCBI SALK_030209C (N670492) = homozygous NA NA

1689678

E-value =

1.15e-34

SALK_092883C (N680004) = homozygous NA NA

>XA_0001

AT1G08820

r Length

VAMP/SYNAPTOBRE

VIN-ASSOCIATED 4e-12 SAIL 278 A07 / Stock: CS812847

SCS2 10813983 PROTEIN 27-2(VAP27- = NA NA Tl

(SAIL_278_A07)

E-value =

2.04e-08 2)

Found in NCBI

Table 2 shows the yeast gene id, the location on the Brassica rapa genome of the Brassica homolog (identified from the Axabidopsis homolog), the best Axabidopsis homologs (including their BLAST score), the name of the Arabidopsis T-DNA knock-out mutants for each Arabidopsis homolog, from the NASC/SALK mutant collection, whether the mutant is heterozygous or homozygous, a phenotype if available and the T- DNA generation of the seed.

_p

75

Genomic sequences for each gene knocked out in each Arabidopsis thaliana mutant is indicated below:

(i) Arabidopsis thaliana CDSl - SEQ ID NO: 54

- Arabidopsis thaliana phosphatidate cytidylyltransferase (CDS 1 )

- other names ATCDSl, CDP-DIACYLGLYCEROL SYNTHASE 1 , CDSl, F2401.17, F2401_17

- encodes protein defined in SEQ ID NO: 49 (NCBI Reference Sequence:

NM_104923.3)

- encoded protien is a CDP-diacylglycerol synthase, involved in phospholipid biosynthesis

- mRNA sequence defined in SEQ ID NO: 44 (NCBI Reference Sequence NMJ04923.3)

(ii) Arabidopsis thaliana CDS2 - SEQ ID NO: 45

- cytidinediphosphate diacylglycerol synthase 2 (CDS2)

- Sequence name AT4G22340.3

- functions in phosphatidate cytidylyltransferase activity

- located in endomembrane system, membrane

- Tair Accession Sequence: 4010726215

(iii) Arabidopsis thaliana RTC2 - SEQ ED NO: 46

- Sequence name AT4G20100.1

- PQ-loop repeat family protein / transmembrane family protein

- Tair accession: Locus: 2119777

(iv) Arabidopsis thaliana Ckbl - SEQ ID NO: 47

- sequence name AT5G47080.1

- Tair Accession Sequence 4010729433

- CASEIN KINASE II BETA CHAIN 1, CKBl, K14A3.3, K14A3J

- Regulatory subunit beta of casein kinase II (CK2) (v) Arabidopsis thaliana SCSI - SEQ ID NO: 48

- Sequence name ATI G08820.1

- Tair Accession Sequence: 1009087352

- encodes VAP33-like protein that interacts with cowpea mosaic virus protein 60K. Is a SNARE-like protein that may be involved in vesicular transport to or from the ER.

Protein sequences identified in Brassica rapa encoded by genes homologous to those knocked out in Arabidopsis are indicated below:

(i) Brassica rapa CDS1 : SEQ ID NO: 1

(ii) Brassica rapa Ckb I : SEQ ID NO: 2 and SEQ ID NO: 3

(iii) Brassica rapa Rtc2: SEQ ID NO: 4

(iv) Brassica rapa Scs2: SEQ ID NO: 5

Gene sequences (egnomic DNA) identified in Brassica rapa homologous to those knocked out in Arabidopsis are indicated below:

(i) Brassica rapa CDS 1 : SEQ ID NO: 50

- Brassica database (http://brassicadb.org) - gene i.d: Bra027046 positions 28523068 28527091 (+ strand)

(ii) Brassica rapa Ckb 1 : SEQ ID NO: 51

- Brassica database (http://brassicadb.org) - gene i.d: Bra025515 positions 8451379 8451925 (- strand)

(iii) Brassica rapa Rtc2: SEQ ID NO: 52

- Brassica database (http://brassicadb.org) - gene i.d: Bra011711 positions 938214 940516 (+ strand)

(iv) Brassica rapa Scs2: SEQ ID NO: 53

Brassica database (http://brassicadb.org) - gene i.d: Bra000978 positions 17997049 17998305 (- strand)

Seeds derived from transformed plants were produced and will be analysed for lipid properties using standard methods. For example, oil bodies may be purified using the method ,

77 described by Tzen et al. (see Tzen et al. (1997), "A new method for seed oil body purification and examination of oil body integrity following germination ", Journal of Biochemistry, 121, 762-768). Briefly, in a typical oil body purification, 100-200 mg of seeds can be soaked in Milli Q grade water for 1 h at room temperature, and subsequently ground 20 times for 15 s in 4 ml of 10 mM sodium phosphate buffer (pH 7.5) containing 0.6 M sucrose (buffer 1) with a Potter grinder driven by a Heidolph motor (rate 7). The sample may be cooled on ice between each grinding cycle, and the potter may be rinsed by 8 ml of buffer 1.

The suspension can be overlaid by one volume of 10 mM sodium phosphate buffer (pH 7.5) containing 0.4 M sucrose (buffer 2) and spun at 10,000 * g and 4 °C for 30 min in a Kontron Ultracentrifuge equipped with a swinging-bucket rotor. The floating oil body fraction can be collected, resuspended in 12 ml of 5 mM sodium phosphate buffer (pH 7.5) containing 0.2 M sucrose and 0.1% (v/v) Tween 20. The suspension can then be overlaid by one volume of 10 mM sodium phosphate buffer (pH 7.5) and spun at 10,000 * g and 4 °C for 30 min in a Kontron Ultracentrifuge equipped with a swinging-bucket rotor. The oil body fraction can be resuspended in 12 ml of buffer 1 additionally containing 2 M NaCl, overlaid by one volume of 10 mM sodium phosphate buffer containing 0.25 M sucrose and 2 M NaCl and spun. The oil body fraction can be collected (CF3), resuspended in 8 ml of 9 M urea and left on a shaker (60 rpm) at room temperature for 10 min. Then the suspension can be placed in centrifuge tubes, overlaid by one volume of 10 mM sodium phosphate buffer (pH 7.5) and spun. The oil body fraction can be collected (CF4), resuspended in 4 ml of buffer 1 and then mixed with 4 ml of hexane. After centrifugation and removal of the upper hexane layer, the oil body fraction can be collected and resuspended in 4 ml of buffer 1. In a last step, the resuspension can be overlaid by one volume of buffer 2 and centrifuged. The floating oil body fraction can be collected (CF6), resuspended in a minimal volume of buffer 1 and stored at 4 °C till further use.

Lipid anaylsis will be performed using standard techniques such as those described in Example 1 above. For example, thin layer chromatography (TLC) may be used (see, for example Fei et al. (2008), "Fldlp, a functional homologue of human seipin, regulates the size of lipid droplets in yeasf, J Cell Biol 180: 473-482; Low et al. (2008), "Caspase-dependent and -independent lipotoxic cell-death pathways in fission yeas f, J Cell Sci 121 : 2671-2684).

It is envisaged that lipid analysis will reveal increased quantities/proportions of triacylglycerol (TAG) production, and in particular increased quantities/proportions of TAGs comprising saturated fatty acid chains.